Compounds for the modulation of beta-catenin expression

ABSTRACT

The invention relates to oligomer compounds (oligomers), which target beta-catenin mRNA in a cell, leading to reduced expression of beta-catenin. Reduction of beta-catenin expression is beneficial for a range of medical disorders, such as hyperproliferative disorders, such as cancer. The invention provides therapeutic compositions comprising oligomers and methods for modulating the expression of beta-catenin using said oligomers, including methods of treatment.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/356,923, filed Jan. 21, 2009, which is a continuation ofU.S. patent application Ser. No. 12/113,031, filed Apr. 30, 2008, nowabandoned, which in turn claims the benefit from U.S. ProvisionalApplication Ser. Nos. 61/023,244, filed Jan. 24, 2008, and 60/915,371,filed May 1, 2007, the disclosure each of which are incorporated hereinby reference in their entireties.

2. FIELD OF THE INVENTION

The invention relates to oligomeric compounds (oligomers) which targetbeta-catenin mRNA in a cell, leading to reduced expression ofbeta-catenin. Reduction of beta-catenin expression is beneficial for arange of medical disorders, such as hyperproliferative diseases,including cancer. The invention provides therapeutic compositionscomprising oligomers and methods for modulating the expression ofbeta-catenin using said oligomers, including methods of treatment.

3. BACKGROUND

Beta-catenin (also known as cadherin-associated protein and (β)-Catenin)is a member of the catenin family of cytosolic proteins and a pivotalplayer in the signalling pathway initiated by Wnt proteins, mediators ofseveral developmental processes. Beta-catenin undergoes phosphorylationupon growth factor stimulation resulting in reduced cell adhesion.

The role of beta-catenin in the development of cancer has been shown tobe regulated by the expression product of the APC (adenomatous polyposisof the colon) gene. The APC tumor suppressor protein binds tobeta-catenin, while beta-catenin was shown to interact with Tcf and Leftranscription factors. Morin et al. (Morin et al., Science, 1997, 275,1787-1790) report that APC protein down-regulates the transcriptionalactivation mediated by beta-catenin and Tcf-4 in colon cancer. Theirresults indicate that the regulation of beta-catenin is critical toAPC's tumor suppressive effect and that this regulation can becircumvented by mutations in either APC or beta-catenin.

Morin et al. showed that mutations of beta-catenin which affectphosphorylation sites rendered the cells insensitive to APC-mediateddown-regulation of beta-catenin and that this disrupted mechanism wascritical to colorectal tumorigenesis (Morin et al., Science, 1997, 275,1787-1790).

Several studies report on the detection of mutations in beta-catenin invarious cancer cell lines and abnormally high amounts of beta-cateninhave been found in melanoma cell lines.

U.S. Pat. No. 6,066,500 to Bennett et al. describes antisense compounds,compositions and methods for modulating the expression of beta-catenin.

Considering the involvement of beta-catenin in the development ofcancer, there remains a need for agents capable of effectivelyinhibiting beta-catenin function.

4. SUMMARY OF THE INVENTION

The invention provides an oligomer of 10-50 contiguous monomers whereinthe sequence of said oligomer is at least 80% identical to the sequenceof the reverse complement of a target region of a nucleic acid whichencodes a mammalian beta-catenin.

The invention provides an oligomer of 10-50 contiguous monomers whichcomprises a first region, wherein the sequence of the first region is atleast 80% identical to the sequence of the reverse complement of atarget region of SEQ ID NO 173 or to a sequence selected from SEQ IDNOs: 1-132, SEQ ID NOs 174-192 and SEQ ID NO: 193.

The invention further provides a conjugate comprising the oligomeraccording to the invention, which, is covalently linked to one or moremoieties that are not themselves nucleic acids or monomers (“conjugatedmoiety”). In some embodiments, the conjugated moiety consists of orcomprises a sterol group such as cholesterol, or other moiety thatfacilitates entry into the cell.

The invention provides for a conjugate comprising the oligomer accordingto the invention, which is covalently linked to a polymeric conjugatedmoiety containing positively charged groups, such as polyethylene glycol(PEG)—i.e. the oligomer according to the invention may, optionally, bepegylated.

The invention provides for pharmaceutical compositions comprising theoligomer or conjugate of the invention, and a pharmaceuticallyacceptable diluent, carrier, salt or adjuvant.

The invention further provides for an oligomer or conjugate according tothe invention, for use in medicine.

The invention further provides for the use of the oligomer or conjugateof the invention for the manufacture of a medicament for the treatmentof one or more of the diseases referred to herein, such as ahyperproliferative disease, e.g., cancer.

The invention further provides for an oligomer or conjugate according tothe invention, for use for the treatment of one or more of the diseasesreferred to herein, such as cancer.

Pharmaceutical and other compositions comprising the oligomer orconjugate of the invention are also provided. Further provided aremethods of down-regulating the expression of beta-catenin in cells ortissues comprising contacting said cells or tissues, in vitro or invivo, with an effective amount of one or more of the oligomers,conjugates or compositions of the invention.

Also disclosed are methods of treating an animal or a human, suspectedof having or being prone to a disease or condition associated withexpression or over-expression of beta-catenin, by administering to saidanimal or human a therapeutically or prophylactically effective amountof one or more of the oligomers, conjugates or compositions of theinvention.

Further, methods of using oligomers for the inhibition of expression ofbeta-catenin, and for treatment of diseases associated with activity ofbeta-catenin are provided.

The invention provides for a method of inhibiting or reducing theexpression of beta-catenin in a cell or a tissue, the method comprisingthe step of contacting said cell or tissue with an effective amount ofan oligomer, a conjugate, or a pharmaceutical composition according tothe invention so that expression of beta-catenin is inhibited orreduced.

The invention provides for a method of triggering apoptosis in a cell,such as a cancer cell, said method comprising the step of contactingsaid cell or tissue with an effective amount of an oligomer, aconjugate, or a pharmaceutical composition according to the invention sothat either expression of beta-catenin is inhibited or reduced and/orapoptosis is triggered.

The invention further provides for an oligomer which comprises orconsists of contiguous covalently linked monomers, wherein the oligomercomprises a first region, wherein the sequence of said first region isat least 80% identical to the sequence of a region of SEQ ID NOs 1-132or SEQ ID NOs 174-193. In some embodiments, the sequence is selectedfrom the group consisting of SEQ ID NO 189, 192, 58 and 103.

The invention further provides for an oligomer which comprises orconsists of a first region, wherein the sequence of said first region isidentical to the sequence of a region of SEQ ID NO 174-193, such as aregion of SEQ ID NO 189 or 192.

The invention further provides for an oligomer which comprises orconsists of a sequence identically present in SEQ ID NO 133-174, such asidentically present in SEQ ID NO 168 or 171.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The beta-catenin sequences that are targeted by the oligomershaving the sequence of SEQ ID NOs: 1, 16, 17, 18, 33, 34, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 73, 88, 103 and 118, respectively, are shownin bold and underlined, indicating their position in the beta-catenintranscript (GenBank Accession number NM_(—)001904—SEQ ID NO: 173).

FIG. 2: Beta-catenin expression normalized to GAPDH 24 hours aftertransfection of SW480 cells.

FIG. 3: Beta-catenin protein content in SW480 cells transfected witholigonucleotides at 1 and 5 nM concentration measured by Western blot.Tubulin staining was used as loading control and SEQ ID NO: 194 was usedas a scrambled control oligonucleotide.

FIG. 4: Cellular proliferation measured using MTS assay in HCT116 cellstransfected with oligonucleotides at 25 nM concentration measured asOD490 at different timepoints after transfection. Mock transfected cellsand cells transfected with SEQ ID NO: 194 (scrambled control) were usedas controls.

FIG. 5: Beta-catenin mRNA expression relative to saline treated controlin mouse liver measured by QPCR after treatment with 3×25 mg/kgoligonucleotide i.v. Beta-catenin expression was normalized to GAPDH andresults were plotted relative to saline treated control.

FIG. 6: The nucleotide sequence of a human beta-catenin mRNA havingAccession No: NM_(—)001904.3 (SEQ ID NO: 195).

6. DETAILED DESCRIPTION The Oligomer

In a first aspect, oligomeric compounds (referred herein as oligomers)are provided that are useful, e.g., in modulating the function ofnucleic acid molecules encoding mammalian beta-catenin, such as thebeta-catenin nucleic acid shown in SEQ ID NO: 173, and naturallyoccurring allelic variants of such nucleic acid molecules encodingmammalian beta-catenin. The oligomers of the invention are composed ofcovalently linked monomers.

The term “monomer” includes both nucleosides and deoxynucleosides(collectively, “nucleosides”) that occur naturally in nucleic acids andthat do not contain either modified sugars or modified nucleobases,i.e., compounds in which a ribose sugar or deoxyribose sugar iscovalently bonded to a naturally-occurring, unmodified, nucleobase(base) moiety (i.e., the purine and pyrimidine heterocycles adenine,guanine, cytosine, thymine or uracil) and “nucleoside analogues”, whichare nucleosides that either do occur naturally in nucleic acids or donot occur naturally in nucleic acids, wherein either the sugar moiety isother than a ribose or a deoxyribose sugar (such as bicyclic sugars or2′ modified sugars, such as 2′ substituted sugars), or the base moietyis modified (e.g., 5-methylcytosine), or both.

An “RNA monomer” is a nucleoside containing a ribose sugar and anunmodified nucleobase.

A “DNA monomer” is a nucleoside containing a deoxyribose sugar and anunmodified nucleobase.

A “Locked Nucleic Acid monomer”, “locked monomer”, or “LNA monomer” is anucleoside analogue having a bicyclic sugar, as further described hereinbelow.

The terms “corresponding nucleoside analogue” and “correspondingnucleoside” indicate that the base moiety in the nucleoside analogue andthe base moiety in the nucleoside are identical. For example, when the“nucleoside” contains a 2-deoxyribose sugar linked to an adenine, the“corresponding nucleoside analogue” contains, for example, a modifiedsugar linked to an adenine base moiety.

The terms “oligomer”, “oligomeric compound”, and “oligonucleotide” areused interchangeably in the context of the invention, and refer to amolecule formed by covalent linkage of two or more contiguous monomersby, for example, a phosphate group (forming a phosphodiester linkagebetween nucleosides) or a phosphorothioate group (forming aphosphothioester linkage between nucleosides). The oligomer consists of,or comprises, 10-50 monomers.

In some embodiments, an oligomer comprises nucleosides, or nucleosideanalogues, or mixtures thereof as referred to herein. An “LNA oligomer”or “LNA oligonucleotide” refers to an oligonucleotide containing one ormore LNA monomers.

Nucleoside analogues that are optionally included within oligomers mayfunction similarly to corresponding nucleosides, or may have specificimproved functions. Oligomers wherein some or all of the monomers arenucleoside analogues are often preferred over native forms because ofseveral desirable properties of such oligomers, such as the ability topenetrate a cell membrane, good resistance to extra- and/orintracellular nucleases, and high affinity and specificity for thenucleic acid target. LNA monomers are particularly preferred, forexample, for conferring several of the above-mentioned properties.

In various embodiments, one or more nucleoside analogues present withinthe oligomer are “silent” or “equivalent” in function to thecorresponding natural nucleoside, i.e. have no functional effect on theway the oligomer functions to inhibit target gene expression. Such“equivalent” nucleoside analogues are nevertheless useful if, forexample, they are easier or cheaper to manufacture, or are more stableunder storage or manufacturing conditions, or can incorporate a tag orlabel. Typically, however, the analogues will have a functional effecton the way in which the oligomer functions to inhibit expression; forexample by producing increased binding affinity to the target region ofthe target nucleic acid and/or increased resistance to intracellularnucleases and/or increased ease of transport into the cell.

Thus, in various embodiments, oligomers according to the inventioncomprise both nucleoside monomers and at least one nucleoside analoguemonomer, such as an LNA monomer, or other nucleoside analogue monomers.

The term “at least one” comprises the integers larger than or equal to1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 and so forth. In various embodiments, such as when referringto the nucleic acid or protein targets of the compounds of theinvention, the term “at least one” includes the terms “at least two” andat “least three” and “at least four”, likewise the term “at least two”may comprise the terms at “least three” and “at least four”.

In various embodiments, the oligomer consists of 10-50 monomers,preferably 10-25 monomers, more preferably 10-16 monomers, and even morepreferably 12-16 monomers.

In various embodiments, the oligomer of the invention does not compriseRNA monomers.

The term “region”, when used to refer to the oligomers of the invention,means a number of contiguous monomers within the oligomer, wherein thenumber of monomers in the “region” is less than the total number ofmonomers in the oligomer.

It is preferred that the oligomers according to the invention are linearmolecules, or are linear as synthesised. The oligomer is, in suchembodiments, a single stranded molecule, and typically does not comprisea short region of: for example, at least 3, 4 or 5 contiguous monomers,which are complementary to another region within the same oligomer suchthat the oligomer forms an internal duplex. In various embodiments, theoligomer is not substantially double-stranded, i.e., is not a siRNA.

In some embodiments, the oligomer of the invention consists of acontiguous stretch of monomers, the sequence of which is identified by aSEQ ID No. disclosed herein (see, e.g., Tables 1-4). In otherembodiments, the oligomer comprises a first region, the regionconsisting of a contiguous stretch of monomers, and one or moreadditional regions which consist of at least one additional monomer. Insome embodiments, the sequence of the first region is identified by aSEQ ID No. disclosed herein.

Target Nucleic Acid

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein, and are defined as a molecule formed by covalent linkage of twoor more monomers, as above-described. Including 2 or more monomers,“nucleic acids” may be of any length, and the term is generic to“oligomers”, which have the lengths described herein. The terms “nucleicacid” and “polynucleotide” include single-stranded, double-stranded,partially double-stranded, and circular molecules.

The term “target nucleic acid”, as used herein, refers to the nucleicacid (such as DNA) encoding mammalian beta-catenin polypeptide, such ashuman beta-catenin protein (e.g., such as the human gene exons havingAccession Nos. X89579, X89593, X89592, X89591, X89588, X89585, X89584and X89578), or a mammalian beta-catenin mRNA such as SEQ ID NO 173.Examples of other mammalian beta-catenin mRNAs include, but are notlimited to, those having Accession Numbers NM_(—)001098203,NM_(—)0010987210 (human beta-catenin mRNA); NM_(—)007614 (mousebeta-catenin mRNA); NM_(—)053357 (rat beta-catenin mRNA); NM-001122762,DQ267491 (horse beta-catenin mRNA); NM_(—)214367 (pig beta-cateninmRNA); BC119949, BT030683 (cow beta-catenin mRNA). “Target nucleic acid”also includes beta-catenin encoding nucleic acids or naturally occurringvariants thereof, and RNA nucleic acids derived therefrom, preferablymRNA, such as pre-mRNA, although preferably mature mRNA. In variousembodiments, for example when used in research or diagnostics, the“target nucleic acid” may be a cDNA or a synthetic oligonucleotidederived from the above DNA or RNA nucleic acid targets. The oligomersaccording to the invention are preferably capable of hybridising to thetarget nucleic acid.

The term “naturally occurring variant thereof’ refers to variants of thebeta-catenin polypeptide or nucleic acid sequence which exist naturallywithin the defined taxonomic group, such as mammalian, such as mouse,monkey, and preferably human. Typically, when referring to “naturallyoccurring variants” of a polynucleotide the term also may encompass anyallelic variant of the beta-catenin-encoding genomic DNA which is foundat the Chromosome Chr 3: 41.22-41.26 Mb by chromosomal translocation orduplication, and the RNA, such as mRNA derived therefrom. Whenreferenced to a specific polypeptide sequence, e.g., the term alsoincludes naturally occurring forms of the protein which may therefore beprocessed, e.g. by co- or post-translational modifications, such assignal peptide cleavage, proteolytic cleavage, glycosylation, etc.

An oligomer of the invention binds to a region of the target nucleicacid (the “target region”) by either Watson-Crick base pairing,Hoogsteen hydrogen bonding, or reversed Hoogsteen hydrogen bonding,between the monomers of the oligomer and monomers of the target nucleicacid. Unless otherwise indicated, binding is by Watson-Crick pairing ofcomplementary bases, and the oligomer binds to the target because thesequence of the oligomer is identical to, or partially-identical to, thesequence of the reverse complement of the target region; for purposesherein, the oligomer is said to be “complementary” or “partiallycomplementary” to the target region, and the percentage of“complementarity” of the oligomer sequence to that of the target regionis the percentage “identity” to the reverse complement of the sequenceof the target region.

Unless otherwise made clear by context, the “target region” herein willbe the region of the target having the sequence that best aligns withthe reverse complement of the sequence of the specified oligomer (orregion thereof), using the alignment program and parameters describedherein below.

In determining the degree of “complementarity” between oligomers of theinvention (or regions thereof) and the target region of the nucleic acidwhich encodes mammalian beta-catenin, such as those disclosed herein,the degree of “complementarity” (also, “homology”) is expressed as thepercentage identity between the sequence of the oligomer (or regionthereof) and the reverse complement of the sequence of the target regionthat best aligns therewith. The percentage is calculated by counting thenumber of aligned bases that are identical as between the 2 sequences,dividing by the total number of contiguous monomers in the oligomer, andmultiplying by 100. In such a comparison, if gaps exist, it ispreferable that such gaps are merely mismatches rather than areas wherethe number of monomers within the gap differ between the oligomer of theinvention and the target region.

Amino acid and polynucleotide alignments, percentage sequence identity,and degree of complementarity may be determined for purposes of theinvention using the ClustalW algorithm using standard settings. Method:EMBOSS::water (local): Gap Open=10.0, Gap extend=0.5, using Blosum 62(protein), or DNAfull for nucleotide/nucleobase sequences.

As will be understood, depending on context, “mismatch” refers to anonidentity in sequence (as, for example, between the nucleobasesequence of an oligomer and the reverse complement of the target regionto which it binds; as for example, between the base sequence of twoaligned beta-catenin encoding nucleic acids), or to noncomplementarityin sequence (as, for example, between an oligomer and the target regionto which binds).

Suitably, the oligomer (or conjugate, as further described, below) iscapable of inhibiting (such as, by down-regulating) expression of thebeta-catenin gene.

In various embodiments, the oligomers of the invention effect inhibitionof beta-catenin in mRNA expression of at least 10% as compared to thenormal expression level, at least 20%, more preferably at least 30%,40%, 50%, 60%, 70%, 80%, 90% or 95% as compared to the normal expressionlevel. In various embodiments, the oligomers of the invention effectinhibition of beta-catenin protein expression of at least 10% ascompared to the normal expression level, at least 20%, more preferablyat least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% as compared to thenormal expression level. In some embodiments, such inhibition is seenwhen using 1 nM of the oligomer or conjugate of the invention. Invarious embodiments, such inhibition is seen when using 25 nM of theoligomer or conjugate.

In various embodiments, the inhibition of mRNA expression is less than100% (i.e., less than complete inhibition of expression), such as lessthan 98%, inhibition, less than 95% inhibition, less than 90%inhibition, less than 80% inhibition, such as less than 70% inhibition.In various embodiments, the inhibition of protein expression is lessthan 100% (i.e., less than complete inhibition of expression), such asless than 98%, inhibition, less than 95% inhibition, less than 90%inhibition, less than 80% inhibition, such as less than 70% inhibition.

Modulation (i.e., inhibition or increase) of expression level may bedetermined by measuring protein levels, e.g. by the methods such asSDS-PAGE followed by western blotting using suitable antibodies raisedagainst the target protein. Alternatively, modulation of expressionlevels can be determined by measuring levels of mRNA, e.g. by northernblotting or quantitative RT-PCR. When measuring via mRNA levels, thelevel of inhibition when using an appropriate dosage, such as 1 and 25nM, is, in various embodiments, typically to a level of 10-20% of thenormal levels in the absence of the compound of the invention.

The invention therefore provides a method of inhibiting (e.g., bydown-regulating) the expression of beta-catenin protein and/or mRNA in acell which is expressing beta-catenin protein and/or mRNA, the methodcomprising contacting the cell with an amount of the oligomer orconjugate according to the invention effective to inhibit (e.g., todown-regulate) the expression of beta-catenin protein and/or mRNA insaid cell. Suitably the cell is a mammalian cell, such as a human cell.The contacting may occur, in certain embodiments, in vitro. In otherembodiments, the contacting may be effected in vivo, by administeringthe compound or conjugate of the invention to a mammal.

An oligomer of the invention typically binds to a target region of thehuman beta-catenin mRNA, and as such, comprises or consists of a regionhaving a base sequence that is complementary or partially complementaryto the base sequence of, e.g., a target region of SEQ ID NO:173. Thesequence of the oligomers of the invention may optionally comprise 1, 2,3, 4 or more base mismatches when compared to the sequence of thebest-aligned target region of SEQ ID NO: 173.

In some embodiments, the oligomers of the invention have sequences thatare identical to a sequence selected from the group consisting of SEQ LDNOS: 1-132, shown in Table 1, below. In other embodiments, the oligomersof the invention have sequences that differ in one, two or three baseswhen compared to a sequence selected from the group consisting of SEQ IDNOs: 1-132. In some embodiments, the oligomers consist of or comprise10-16 contiguous monomers. Examples of oligomers consisting of 16contiguous monomers are SEQ ID NOS: 1, 16, 17, 18, 33, 34, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 73, 88, 103, and 118. Shorter sequences canbe derived therefrom, e.g., the sequence of the shorter oligomer may beidentically present in a region of SEQ ID NOs: 1-132. Longer oligomersmay include a region having a sequence of at least 10 contiguousmonomers that is identically present in SEQ ID NOs: 1-132.

TABLE 1 Antisense oligonucleotide sequences (Motifs) Sequence  LengthSEQ ID NO (5′-3′) (bases) Description SEQ ID NO: 1 GAAAGCTGATGGACC  16Antisense  A oligo sequence SEQ ID NO: 2 GAAAGCTGATGGACC 15 as aboveSEQ ID NO: 3 AAAGCTGATGGACCA 15 as above SEQ ID NO: 4 GAAAGCTGATGGAC 14as above SEQ ID NO: 5 AAAGCTGATGGACC 14 as above SEQ ID NO: 6AAGCTGATGGACCA 14 as above SEQ ID NO: 7 GAAAGCTGATGGA 13 as aboveSEQ ID NO: 8 AAAGCTGATGGAC 13 as above SEQ ID NO: 9 AAGCTGATGGACC 13as above SEQ ID NO: 10 AGCTGATGGACCA 13 as above SEQ ID NO: 11GAAAGCTGATGG 12 as above SEQ ID NO: 12 AAAGCTGATGGA 12 as aboveSEQ ID NO: 13 AAGCTGATGGAC 12 as above SEQ ID NO: 14 AGCTGATGGACC 12as above SEQ ID NO: 15 GCTGATGGACCA 12 as above SEQ ID NO: 16CAGACTTAAAGATGGC 16 as above SEQ ID NO: 17 CAGAATCCACTGGTGA 16 as aboveSEQ ID NO: 18 GCACTGCCATTTTAGC 16 as above SEQ ID NO: 19 GCACTGCCATTTTAG15 as above SEQ ID NO: 20 CACTGCCATTTTAGC 15 as above SEQ ID NO: 21GCACTGCCATTTTA 14 as above SEQ ID NO: 22 CACTGCCATTTTAG 14 as aboveSEQ ID NO: 23 ACTGCCATTTTAGC 14 as above SEQ ID NO: 24 GCACTGCCATTTT 13as above SEQ ID NO: 25 CACTGCCATTTTA 13 as above SEQ ID NO: 26ACTGCCATTTTAG 13 as above SEQ ID NO: 27 CTGCCATTTTAGC 13 as aboveSEQ ID NO: 28 GCACTGCCATTT 12 as above SEQ ID NO: 29 CACTGCCATTTT 12as above SEQ ID NO: 30 ACTGCCATTTTA 12 as above SEQ ID NO: 31CTGCCATTTTAG 12 as above SEQ ID NO: 32 TGCCATTTTAGC 12 as aboveSEQ ID NO: 33 GTAATAGCCAAGAATT 16 as above SEQ ID NO: 34ACTCTGCTTGTGGTCC 16 as above SEQ ID NO: 35 ACTCTGCTTGTGGTC 15 as aboveSEQ ID NO: 36 CTCTGCTTGTGGTCC 15 as above SEQ ID NO: 37 ACTCTGCTTGTGGT14 as above SEQ ID NO: 38 CTCTGCTTGTGGTC 14 as above SEQ ID NO: 39TCTGCTTGTGGTCC 14 as above SEQ ID NO: 40 ACTCTGCTTGTGG 13 as aboveSEQ ID NO: 41 CTCTGCTTGTGGT 13 as above SEQ ID NO: 42 TCTGCTTGTGGTC 13as above SEQ ID NO: 43 CTGCTTGTGGTCC 13 as above SEQ ID NO: 44ACTCTGCTTGTG 12 as above SEQ ID NO: 45 CTCTGCTTGTGG 12 as aboveSEQ ID NO: 46 TCTGCTTGTGGT 12 as above SEQ ID NO: 47 CTGCTTGTGGTC 12as above SEQ ID NO: 48 TGCTTGTGGTCC 12 as above SEQ ID NO: 49CCACCAGCTTCTACAA 16 as above SEQ ID NO: 50 GAGTCCAAAGACAGTT 16 as aboveSEQ ID NO: 51 ACCCACTTGGCAGACC 16 as above SEQ ID NO: 52GCACAAACAATGGAAT 16 as above SEQ ID NO: 53 GCAGCTACTCTTTGGA 16 as aboveSEQ ID NO: 54 CTCCCTCAGCTTCAAT 16 as above SEQ ID NO: 55GCAGTCTCATTCCAAG 16 as above SEQ ID NO: 56 TATCCACCAGAGTGAA 16 as aboveSEQ ID NO: 57 CATCCATGAGGTCCTG 16 as above SEQ ID NO: 58CCATCTTGTGATCCAT 16 as above SEQ ID NO: 59 CCATCTTGTGATCCA 15 as aboveSEQ ID NO: 60 CATCTTGTGATCCAT 15 as above SEQ ID NO: 61 CCATCTTGTGATCC14 as above SEQ ID NO: 62 CATCTTGTGATCCA 14 as above SEQ ID NO: 63ATCTTGTGATCCAT 14 as above SEQ ID NO: 64 CCATCTTGTGATC 13 as aboveSEQ ID NO: 65 CATCTTGTGATCC 13 as above SEQ ID NO: 66 ATCTTGTGATCCA 13as above SEQ ID NO: 67 TCTTGTGATCCAT 13 as above SEQ ID NO: 68CCATCTTGTGAT 12 as above SEQ ID NO: 69 CATCTTGTGATC 12 as aboveSEQ ID NO: 70 ATCTTGTGATCC 12 as above SEQ ID NO: 71 TCTTGTGATCCA 12as above SEQ ID NO: 72 CTTGTGATCCAT 12 as above SEQ ID NO: 73AAGCAAGCAAAGTCAG 16 as above SEQ ID NO: 74 AAGCAAGCAAAGTCA 15 as aboveSEQ ID NO: 75 AGCAAGCAAAGTCAG 15 as above SEQ ID NO: 76 AAGCAAGCAAAGTC14 as above SEQ ID NO: 77 AGCAAGCAAAGTC 13 as above SEQ ID NO: 78GCAAGCAAAGTCAG 14 as above SEQ ID NO: 79 AAGCAAGCAAAGT 13 as aboveSEQ ID NO: 80 AGCAAGCAAAGTC 13 as above SEQ ID NO: 81 GCAAGCAAAGTCA 13as above SEQ ID NO: 82 CAAGCAAAGTCAG 13 as above SEQ ID NO: 83AAGCAAGCAAAG 12 as above SEQ ID NO: 84 AGCAAGCAAAGT 12 as aboveSEQ ID NO: 85 GCAAGCAAAGTC 12 as above SEQ ID NO: 86 CAAGCAAAGTCA 12as above SEQ ID NO: 87 AAGCAAAGTCAG 12 as above SEQ ID NO: 88GAAATTGCTGTAGCAG 16 as above SEQ ID NO: 89 GAAATTGCTGTAGCA 15 as aboveSEQ ID NO: 90 AAATTGCTGTAGCAG 15 as above SEQ ID NO: 91 GAAATTGCTGTAGC14 as above SEQ ID NO: 92 AAATTGCTGTAGCA 14 as above SEQ ID NO: 93AATTGCTGTAGCAG 14 as above SEQ ID NO: 94 GAAATTGCTGTAG 13 as aboveSEQ ID NO: 95 AAATTGCTGTAGC 13 as above SEQ ID NO: 96 AATTGCTGTAGCA 13as above SEQ ID NO: 97 ATTGCTGTAGCAG 13 as above SEQ ID NO: 98GAAATTGCTGTA 12 as above SEQ ID NO: 99 AAATTGCTGTAG 12 as aboveSEQ ID NO: 100 AATTGCTGTAGC 12 as above SEQ ID NO: 101 ATTGCTGTAGCA 12as above SEQ ID NO: 102 TTGCTGTAGCAG 12 as above SEQ ID NO: 103GTGTTCTACACCATTA 16 as above SEQ ID NO: 104 GTGTTCTACACCATT 15 as aboveSEQ ID NO: 105 TGTTCTACACCATTA 15 as above SEQ ID NO: 106 GTGTTCTACACCAT14 as above SEQ ID NO: 107 TGTTCTACACCATT 14 as above SEQ ID NO: 108GTTCTACACCATTA 14 as above SEQ ID NO: 109 GTGTTCTACACCA 13 as aboveSEQ ID NO: 110 TGTTCTACACCAT 13 as above SEQ ID NO: 111 GTTCTACACCATT 13as above SEQ ID NO: 112 TTCTACACCATTA 13 as above SEQ ID NO: 113GTGTTCTACACC 12 as above SEQ ID NO: 114 TGTTCTACACCA 12 as aboveSEQ ID NO: 115 GTTCTACACCAT 12 as above SEQ ID NO: 116 TTCTACACCATT 12as above SEQ ID NO: 117 TCTACACCATTA 12 as above SEQ ID NO: 118AACATGAAATAGATCC 16 as above SEQ ID NO: 119 AACATGAAATAGATC 15 as aboveSEQ ID NO: 120 ACATGAAATAGATCC 15 as above SEQ ID NO: 121 AACATGAAATAGAT14 as above SEQ ID NO: 122 ACATGAAATAGATC 14 as above SEQ ID NO: 123CATGAAATAGATCC 14 as above SEQ ID NO: 124 AACATGAAATAGA 13 as aboveSEQ ID NO: 125 ACATGAAATAGAT 13 as above SEQ ID NO: 126 CATGAAATAGATC 13as above SEQ ID NO: 127 ATGAAATAGATCC 13 as above SEQ ID NO: 128AACATGAAATAG 12 as above SEQ ID NO: 129 ACATGAAATAGA 12 as aboveSEQ ID NO: 130 CATGAAATAGAT 12 as above SEQ ID NO: 131 ATGAAATAGATC 12as above SEQ ID NO: 132 TGAAATAGATCC 12 as above

Further provided are target nucleic acids (i.e., DNA or mRNA encodingbeta-catenin), that contain target regions that are complementary orpartially-complementary to one or more of the oligomers of SEQ ID NOs1-132, wherein said oligomers are capable of inhibiting expression(e.g., by down-regulation) of beta-cat en in protein or mRNA. Forexample, target regions of human beta-catenin mRNA which arecomplementary to the antisense oligomers having the sequences of SEQ IDNOs: 1, 16, 17, 18, 33, 34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 73,88, 103 and 118, are shown in FIG. 1 (bold and underlined, with thecorresponding oligomer SEQ ID NOs indicated above).

The oligomer of the invention may, suitably, comprise a region having aparticular sequence, such as an oligomer selected from SEQ ID NOS:174-193, that is identically present in a shorter oligomer of theinvention. Preferably, the region comprises 10-16 monomers. For example,SEQ ID NOs: 174-193 each comprise a region wherein the sequence of theregion is identically present in the shorter oligomers having SEQ IDNOS: 1, 16, 17, 18, 33, 34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 73,88, 103, and 118, respectively. In some embodiments, oligomers whichhave fewer than 16 monomers, such as 10, 11, 12, 13, 14, or 15, have aregion of at least 8, at least 9, at least 10, at least 11, at least 12,at least 13, at least 14 or 15, contiguous monomers wherein the sequenceof the region is identically present in SEQ ID NOS: 1, 16, 17, 18, 33,34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 73, 88, 103, or 118. Hence,in various embodiments, shorter oligomers are derived from longeroligomers. In some embodiments, longer oligomers include all, or atleast 10 contiguous monomers, from those exemplified SEQ ID NOs.Typically, if an oligomer of the invention comprises a first regionhaving a sequence that is identically present in SEQ ID NOS: 1, 16, 17,18, 33, 34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 73, 88, 103, or 118,and the oligomer is longer than the first region, then the other regionsof the oligomer preferably flank the first region that is identicallypresent in SEQ ID NOS: 1, 16, 17, 18, 33, 34, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 73, 88, 103, or 118, the flanking regions of theoligomer having sequences that are complementary to the sequencesflanking the target region of the target nucleic acid. Two sucholigomers are SEQ ID NO 58 and SEQ ID NO 103.

Specific designs of oligomers of the invention are also disclosed, forexample those shown in SEQ ID NOS: 133-152, in particular SEQ ID NOs:133, 134, 135, 136, 138, 141, 148, 149, 150, 151 and 152. Specificdesigns of LNA oligonucleotides are also disclosed, for example thoseshown in SEQ ID NOS: 153-172, in particular SEQ ID NOS: 153, 154, 155,156, 158, 161, 168, 169, 170, 171 and 172. The oligomers of theinvention are, in preferred embodiments, potent inhibitors ofbeta-catenin mRNA and protein expression.

In various embodiments, the oligomer comprises or consists of a sequenceof monomers which is fully complementary (perfectly complementary) to atarget region of a target nucleic acid which encodes a mammalianbeta-catenin.

However, in some embodiments, the sequence of the oligomer includes 1,2, 3, or 4 (or more) mismatches, as compared to the best-aligned targetregion, and still sufficiently binds to the target region to effectinhibition of beta-catenin mRNA or protein expression. The destabilizingeffect of mismatches on the Watson-Crick hydrogen-bonded duplex may, forexample, be compensated by increased length of the oligomer and/or anincreased number of nucleoside analogues, such as LNA monomers, presentwithin the oligomer.

In various embodiments, the oligomer base sequence comprises no morethan 3, such as no more than 2, mismatches compared to the base sequenceof the best-aligned target region of, for example, a nucleic acid whichencodes a mammalian beta-catenin. In various embodiments, the oligomerbase sequence comprises no more than a single mismatch compared to thebase sequence of the best-aligned target region of, for example, anucleic acid which encodes a mammalian beta-catenin.

The base sequences of the oligomers of the invention or of a regionthereof are preferably at least 80% identical to a sequence selectedfrom the group consisting of SEQ ID NOS: 1-132, such as at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, even100% identical.

The base sequences of the oligomers of the invention or of a regionthereof are preferably at least 80% complementary to a sequence of atarget region present in SEQ ID NO: 173, such as at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, even 100%complementary.

In various embodiments, the sequence of the oligomer (or of a firstregion thereof) is selected from the group consisting of SEQ ID NOS:1-132 or SEQ ID NOS: 174-193, or is selected from the group consistingof at least 10 contiguous monomers of SEQ ID NOS: 1-132 or SEQ ID NOS:174-193. In another embodiment, the sequence of the oligomer of theinvention or first region thereof optionally comprises 1, 2, or 3 basemoieties that differ from those in SEQ ID NOs: 1-132 or SEQ ID NOs:174-193, or at least 10 contiguous monomers thereof, when optimallyaligned with said selected sequence or region thereof.

In certain embodiments, the monomer region consists of 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 contiguousmonomers, such as 10-15, 12 25, 12-22, such as 12-18 monomers. Suitably,in various embodiments, the region is of the same length as the oligomerof the invention.

In some embodiments, the oligomer comprises additional monomers at the5′ or 3′ ends, such as, independently, 1, 2, 3, 4 or 5 additionalmonomers at the 5′ end and/or the 3′ end of the oligomer, which arenon-complementary to the sequence of the target region. In variousembodiments, the oligomer of the invention comprises a region that iscomplementary to the target, which is flanked 5′ and/or 3′ by additionalmonomers. In various embodiments, the 3′ end of the region is flanked by1, 2 or 3 DNA or RNA monomers. 3′ DNA monomers are frequently usedduring solid state synthesis of oligomers. In various embodiments, whichmay be the same or different, the 5′ end of the oligomer is flanked by1, 2 or 3 DNA or RNA monomers. In certain embodiments, the additional 5′or 3′ monomers are nucleosides, such as DNA or RNA monomers. In variousembodiments, the additional 5′ or 3′ monomers may represent region D asreferred to in the context of gapmer oligomers herein.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 1, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 16, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 17, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 18, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:33, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:34, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:49, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:50, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:51, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:52, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:53, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:54, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:55, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:56, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:57, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:58, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:73, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:88, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 103, or according to a region thereof.

In certain embodiments, the oligomer according to the invention consistsof, or comprises, contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:118, or according to a region thereof.

Sequence alignments can be used to identify regions of the nucleic acidsencoding beta-catenin from human and one or more different mammalianspecies, such as monkey, mouse and/or rat, where there are sufficientstretches of nucleic acid identity between or among the species to allowthe design of oligonucleotides which target (that is, which bind withsufficient specificity to inhibit expression of) both the humanbeta-catenin target nucleic acid and the corresponding nucleic acidspresent in the different mammalian species.

In some embodiments, such oligomers consist of or comprise regions of atleast 10, such as at least 12, such as at least 14, such as at least 16,such as at least 18, such as 11, 12, 13, 14, 15, 16, 17 or 18 contiguousmonomers which are 100% complementary in sequence to the sequence of thetarget regions of both the nucleic acid encoding beta-catenin fromhumans and of the nucleic acid(s) encoding beta-catenin from a differentmammalian species.

In some embodiments, the oligomer of the invention comprises or consistsof a region of contiguous monomers having a sequence that is at least80%, such as at least 85%, such as at least 90%, such as at least 95%,such as at least 98%, or 100% complementary to the sequence of thetarget regions of both the nucleic acid encoding human beta-catenin anda nucleic acid(s) encoding beta-catenin from a different mammalianspecies, such as the mouse nucleic acid encoding beta-catenin. It ispreferable that the contiguous nucleobase sequence of the oligomer is100% complementary to the target region of the human beta-catenin mRNA.

Length

The oligomer comprises or consists of 10-50 contiguous monomers, such as10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 contiguous monomers.

In various embodiments, the oligomers comprise or consist of 10-25contiguous monomers, 12-25 or 10-22 contiguous monomers, such as 12-18contiguous monomers, such as 13-17 or 12-16 contiguous monomers, such as13, 14, 15, 16 contiguous monomers.

In various embodiments, the oligomers comprise or consist of 10, 11, 12,13, or 14 contiguous monomers.

In various embodiments, the oligomer according to the invention consistsof no more than 22 contiguous monomers, such as no more than 20contiguous monomers, such as no more than 18 contiguous monomers, suchas 15, 16 or 17 contiguous monomers. In certain embodiments, theoligomer of the invention comprises less than 20 contiguous monomers.

Nucleosides and Nucleoside Analogues

In various embodiments, at least one of the monomers present in theoligomer is a nucleoside analogue that contains a modified base, such asa base selected from the group consisting of 5-methylcytosine,isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil,6-aminopurine, 2-aminopurine, inosine, diaminopurine, and2-chloro-6-aminopurine, xanthine, hypoxanthine, 5-methylcytosine,isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil,6-aminopurine, 2-aminopurine, inosine, diaminopurine, and2-chloro-6-aminopurine.

In various embodiments, at least one of the monomers present in theoligomer is a nucleoside analogue that contains a modified sugar.

In some embodiments, the linkage between at least 2 contiguous monomersof the oligomer is other than a phosphodiester bond.

In certain embodiments, the oligomer includes at least one monomer thathas a modified base, at least one monomer (which may be the samemonomer) having a modified sugar, and at least one inter-monomer linkagethat is non-naturally occurring.

Specific examples of nucleoside analogues useful in the oligomersdescribed herein are described by e.g. Freier & Altmann; Nucl. AcidRes., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in DrugDevelopment, 2000, 3(2), 293-213, and in Scheme 1 (in which somenucleoside analogues are shown as nucleotides):

Scheme 1

The oligomer may thus comprise or consist of a simple sequence ofnucleosides—preferably DNA monomers, but also possibly RNA monomers, ora combination of nucleosides and one or more nucleoside analogues. Insome embodiments, such nucleoside analogues suitably enhance theaffinity of the oligomer for the target region of the target nucleicacid.

Examples of suitable and preferred nucleoside analogues are described inWO 2007/031091, incorporated herein by reference in its entirety, or arereferenced therein.

In some embodiments, the nucleoside analogue comprises a sugar moietymodified to provide a 2′-substituent group, such as 2′-O-alkyl-ribosesugars, 2′-amino-deoxyribose sugars, and 2′-fluoro-deoxyribose sugars.

In some embodiments, the nucleoside analogue comprises a sugar in whicha bridged structure, creating a bicyclic sugar (LNA), is present, whichenhances binding affinity and may also provide some increased nucleaseresistance. In various embodiments, the LNA monomer is selected fromoxy-LNA (such as beta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA(such as beta-D-amino-LNA and alpha-L-amino-LNA) and/or thio-LNA (suchas beta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENAand alpha-L-ENA). In certain embodiments, the LNA monomers arebeta-D-oxy-LNA. LNA monomers are further described, below.

Incorporation of affinity-enhancing nucleoside analogues in theoligomer, such as LNA monomers or monomers containing 2′-substitutedsugars, can allow the size of the oligomer to be reduced, and may alsoreduce the upper limit to the size of the oligomer before non-specificor aberrant binding takes place.

In certain embodiments, the oligomer comprises at least 2 nucleosideanalogues. In some embodiments, the oligomer comprises from 3-8nucleoside analogues, e.g. 6 or 7 nucleoside analogues. In preferredembodiments, at least one of the nucleoside analogues is a lockednucleic acid (LNA) monomer; for example at least 3 or at least 4, or atleast 5, or at least 6, or at least 7, or 8, of the nucleoside analoguesare LNA monomers. In some embodiments all the nucleoside analogues areLNA monomers.

It will be recognised that when referring to a preferred oligomer basesequence, in certain embodiments the oligomers comprise a correspondingnucleoside analogue, such as a corresponding LNA monomer or othercorresponding nucleoside analogue, which raise the duplex stability (Tm)of the oligomer/target region duplex (i.e. affinity enhancing nucleosideanalogues).

In various preferred embodiments, any mismatches (that is,noncomplementarities) between the base sequence of the oligomer and thebase sequence of the target region, if present, are located other thanin the regions of the oligomer that contain affinity-enhancingnucleoside analogues (e.g., regions A or C), such as within region B asreferred to herein below, and/or within region D as referred to hereinbelow, and/or in regions of the oligomer containing only nucleosides,and/or in regions which are 5′ or 3′ to the region of the oligomer thatis complementary to the target region.

In some embodiments the nucleoside analogues present within the oligomerof the invention (such as in regions A and C mentioned herein) areindependently selected from, for example: monomers containing2′-O-alkyl-ribose sugars, monomers containing 2′-aminodeoxyribosesugars, monomers containing 2′-fluoro-deoxyribose sugars, LNA monomers,monomers containing arabinose sugars (“ANA monomers”), monomerscontaining 2′-fluoro-ANA sugars, monomers containing d-arabino-hexitolsugars (“HNA monomers”), intercalating monomers as defined inChristensen, Nucl. Acids. Res. 30: 4918-4925 (2002), hereby incorporatedby reference, and monomers containing 2′MOE sugars. In certainembodiments, there is only one of the above types of nucleosideanalogues present in the oligomer of the invention, or region thereof.

In certain embodiments, the nucleoside analogues contain2′-O-methoxyethyl-ribose sugars (2′MOE), or 2′-fluoro-deoxyribose sugarsor LNA sugars, and as such the oligonucleotide of the invention maycomprise nucleoside analogues which are independently selected fromthese three types of analogue, or may comprise only one type of analogueselected from the three types. In certain oligomer embodimentscontaining nucleoside analogues, at least one of the nucleosideanalogues contains a 2′-MOE-ribose sugar, such as 2, 3, 4, 5, 6, 7, 8, 9or 10 nucleoside analogues containing 2′-MOE-ribose sugars. In certainembodiments, at least one of the nucleoside analogues contains a2′-fluoro deoxyribose sugar, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10nucleoside analogues containing 2′-fluoro-deoxyribose sugars.

In various embodiments, the oligomer according to the inventioncomprises at least one Locked Nucleic Acid (LNA) monomer, such as 1, 2,3, 4, 5, 6, 7, or 8 LNA monomers, such as 3-7 or 4-8 LNA monomers, or 3,4, 5, 6 or 7 LNA monomers. In various embodiments, all of the nucleosideanalogues are LNA monomers. In some embodiments, the oligomer comprisesboth beta-D-oxy-LNA monomers, and one or more of the following LNAunits: thio-LNA monomers, amino-LNA monomers, oxy-LNA monomers, and/orENA monomers in either the beta-D or alpha-L configuration orcombinations thereof. In certain embodiments, the cytosine base moietiesof all LNA monomers in the oligomer are 5-methylcytosines. In certainembodiments, of the invention, the oligomer comprises both LNA and DNAmonomers. Typically, the combined total of LNA and DNA monomers is10-25, preferably 10-20, even more preferably 12-16. In certainembodiments, of the invention, the oligomer, or a region thereof,consists of at least one LNA monomer, and the remaining monomers are DNAmonomers. In certain embodiments, the oligomer comprises only LNAmonomers and nucleosides (such as RNA or DNA monomers, most preferablyDNA monomers), optionally linked with modified linkage groups such asphosphorothioate.

In various embodiments, at least one of the nucleoside analogues presentin the oligomer has a modified base selected from the group consistingof 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine.

LNA

The term “LNA monomer” refers to a nucleoside analogue containing abicyclic sugar (an “LNA sugar”). The terms “LNA oligonucleotide” and“LNA oligomer” refer to an oligomer containing one or more LNA monomers.

The LNA monomer used in the oligomers of the invention preferably hasthe structure of the general formula I:

wherein X is selected from -0-, —S—, —N(RN*)—, —C(R6R6*)-;

B is selected from hydrogen, optionally substituted C1-4-alkoxy,optionally substituted C1-4-alkyl, optionally substituted C1-4-acyloxy,nucleobases, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands;

P designates the radical position for an internucleoside linkage to asucceeding monomer, or a 5′-terminal group, such internucleoside linkageor 5′-terminal group optionally including the substituent R5 or equallyapplicable the substituent R5*;

P* designates an internucleoside linkage to a preceding monomer, or a3′-terminal group;

R4* and R2* together designate a biradical consisting of 1-4groups/atoms selected from —C(RaRb)—, —C(Ra)=C(Rb), —C(Ra)=N, O,—Si(Ra)2-, S—, —So2-, —N(Ra)—, and >C=Z, wherein Z is selected from —O—,—S—, and —N(Ra)—, and Ra and Rb each is independently selected fromhydrogen, optionally substituted C1-12-alkyl, optionally substitutedC2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy,C1-12-alkoxy, C2-12-alkoxyalkyl, C2-12-alkenyloxy, carboxy,C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl,aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl,hetero-aryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C16-alkyl)amino, carbamoyl, mono- anddi(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- anddi(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino,carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro,azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents Ra and Rbtogether may designate optionally substituted methylene (═CH2), and

each of the substituents R1*, R2, R3, R5, R5*, R6 and R6*, which arepresent is independently selected from hydrogen, optionally substitutedC1-12-alkyl, optionally substituted C2-12-alkenyl, optionallysubstituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkoxyalkyl,C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl,formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl,hetero-aryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C1-6-alkyl)amino, carbamoyl, mono- anddi(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- anddi(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino,carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro,azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators,photochemically active groups, thermo chemically active groups,chelating groups, reporter groups, and ligands, where aryl andheteroaryl may be optionally substituted, and where two geminalsubstituents together may designate oxo, thioxo, imino, or optionallysubstituted methylene, or together may form a spiro biradical consistingof a 1-5 carbon atom(s) alkylene chain which is optionally interruptedand/or terminated by one or more heteroatoms/groups selected from O, S,and —(NRN)— where RN is selected from hydrogen and C1-4-alkyl, and wheretwo adjacent (non-geminal) substituents may designate an additional bondresulting in a double bond; and RN*, when present and not involved in abiradical, is selected from hydrogen and C14-alkyl; and basic salts andacid addition salts thereof;

In certain embodiments, R5* is selected from H, —CH3, —CH2-CH3,—CH2-O—CH3, and CH═CH2.

In various embodiments, R4* and R2* together designate a biradicalselected from —C(RaRb)—O, —C(RaRb)—C(RcRd)—O,—C(RaRb)—C(RcRd)—C(ReRf)—O, —C(RaRb)—O—C(RcRd)—, —C(RaRb)—O—C(RcRd)—O,—C(RaRb)—C(RcRd)—, —C(RaRb)—C(RcRd)—C(ReRf)-1, —C(Ra)=C(Rb) C(RcRd)—,—C(RaRb)—N(Rc)—, —C(RaRb)—C(RcRd)-N(Re)—, —C(RaRb)—N(Rc)-0, and—C(RaRb)-S, —C(RaRb)—C(RcRd)-S—, wherein Ra, Rb, Rc, Rd, Re, and Rf eachis independently selected from hydro-gen, optionally substitutedC1-12-alkyl, optionally substituted C2-12-alkenyl, optionallysubstituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkoxyalkyl,C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl,formyl, aryl, aryl-oxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,hetero-aryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C1-6-alkyl)amino, carbamoyl, mono- anddi(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- anddi(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino,carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro,azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents Ra and Rbtogether may designate optionally substituted methylene (═CH2),

In a further embodiment, R4* and R2* together designate a biradicalselected from —CH2-O—,

—CH2-S—, —CH2-NH—, —CH2-N(CH3)-, —CH2-CH2-O—, —CH2-CH(CH3)-,—CH2-CH2-S—, —CH2-CH2-NH—, —CH2-CH2-CH2-, —CH2-CH2-CH2-O—,—CH2-CH2-CH(CH3)-, —CH═CH—CH2-, —CH2-O—CH2-O—, —CH2-NH—O—,—CH2-N(CH3)-O—, —CH2-O—CH2-, —CH(CH3)-O—, —CH(CH2-O—CH3)-O—.

For all chiral centers, asymmetric groups may be found in either R or Sorientation.

Preferably, the LNA monomer used in the oligomers of the inventioncomprises at least one LNA monomer according to any of the formulas

wherein Y is —O—, —O—CH2-, —S—, —NH—, or N(RH); Z and Z* areindependently selected among an internucleoside linkage, a terminalgroup or a protecting group; B constitutes an unmodified base moiety ora modified base moiety that either occurs naturally in nucleic acids ordoes not occur naturally in nucleic acids, and RH is selected fromhydrogen and C1-4-alkyl.

Specifically preferred LNA monomers are shown in Scheme 2:

The term “thio-LNA” refers to an LNA monomer in which Y in the generalformula above is selected from S or —CH2-S—. Thio-LNA can be in eitherthe beta-D or alpha-L-configuration.

The term “amino-LNA” refers to an LNA monomer in which Y in the generalformula above is selected from —N(H)—, N(R)—, CH2-N(H)—, and —CH2-N(R)—where R is selected from hydrogen and C1-4-alkyl. Amino-LNA can be ineither the beta-D or the alpha-L-configuration.

The term “oxy-LNA” refers 10 an LNA monomer in which Y in the generalformula above represents —O— or —CH2-O—. Oxy-LNA can be in either thebeta-D or the alpha-L-configuration.

The term “ENA” refers to an LNA monomer in which Y in the generalformula above is —CH2-O— (where the oxygen atom of —CH2-O— is attachedto the 2′-position relative to the base B).

In a preferred embodiment, the LNA monomer is selected from abeta-D-oxy-LNA monomer, an alpha-L-oxy-LNA monomer, a beta-D-amino-LNAmonomer and a beta-D-thio-LNA monomer, in particular a beta-D-oxy-LNAmonomer.

In the present context, the term “C1-4-alkyl” means a linear or branchedsaturated hydrocarbon chain wherein the chain has from one to fourcarbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl and tert-butyl.

RNAse H Recruitment

It is recognised that an oligomer may function via non-RNase-mediateddegradation of a target mRNA, such as by steric hindrance oftranslation, or other mechanisms; however, the preferred oligomers ofthe invention are capable of recruiting an endoribonuclease (RNase),such as RNase H.

Typically, the oligomer comprises a region of at least 6, such as atleast 7, contiguous monomers, such as at least 8 or at least 9contiguous monomers, including 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16contiguous monomers, which, when forming a duplex with the target regionof the target RNA, is capable of recruiting RNase. The region of theoligomer which is capable of recruiting RNAse may be region B, asreferred to in the context of a gapmer as described herein below. Incertain embodiments, the region of the oligomer which is capable ofrecruiting RNAse, such as region B, consists of 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 monomers.

EP 1 222 309 provides in vitro methods for determining RNaseH activity,which may be used to determine the ability of the oligomers of theinvention to recruit RNaseH. An oligomer is deemed capable of recruitingRNase H if, when contacted with the complementary target region of theRNA target, it has an initial rate, as measured in pmol/l/min, of atleast 1%, such as at least 5%, such as at least 10% or less than 20% ofan oligonucleotide having the same base sequence but containing only DNAmonomers, with no 2′ substitutions, with phosphorothioate linkage groupsbetween all monomers in the oligonucleotide, using the methodologyprovided by Example 91-95 of EP 1 222 309, incorporated herein byreference.

In various embodiments, an oligomer is deemed essentially incapable ofrecruiting RNaseH if, when contacted with the complementary targetregion of the RNA target, and RNaseH, the RNaseH initial rate, asmeasured in pmol/l/min, is less than 1%, such as less than 5%, such asless than 10% or less than 20% of the initial rate determined using anoligonucleotide having the same base sequence, but containing only DNAmonomers, with no 2′ substitutions, with phosphorothioate linkage groupsbetween all monomers in the oligonucleotide, using the methodologyprovided by Example 91-95 of EP 1 222 309.

In other embodiments, an oligomer is deemed capable of recruiting RNaseHif, when contacted with the complementary target region of the RNAtarget, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min,is at least 20%, such as at least 40%, such as at least 60%, such as atleast 80% of the initial rate determined using an oligonucleotide havingthe same base sequence, but containing only DNA monomers, with no 2′substitutions, with phosphorothioate linkage groups between all monomersin the oligonucleotide, using the methodology provided by Example 91-95of EP 1 222 309.

Typically, the region of the oligomer that forms a duplex with thecomplementary target region of the target RNA and is capable ofrecruiting RNase may contain DNA monomers and LNA monomers and may forma DNA/RNA like duplex with the target region. The LNA monomers arepreferably in the alpha-L configuration, particularly preferred beingalpha-L-oxy LNA.

The oligomer of the invention may comprise both nucleosides andnucleoside analogues, and may be in the form of a gapmer, a headmer or amixmer.

A “headmer” is defined as an oligomer that comprises a first region anda second region that is contiguous thereto, with the 5′-most monomer ofthe second region linked to the 3′-most monomer of the first region. Thefirst region comprises a contiguous stretch of non-RNase-recruitingnucleoside analogues, and the second region comprises a contiguousstretch (such as at least 7 contiguous monomers) of DNA monomers ornucleoside analogue monomers recognizable and cleavable by the RNAse.

A “tailmer” is defined as an oligomer that comprises a first region anda second region that is contiguous thereto, with the 5′-most monomer ofthe second region linked to the 3′-most monomer of the first region. Thefirst region comprises a contiguous stretch (such as at least 7 suchmonomers) of DNA monomers or nucleoside analogue monomers recognizableand cleavable by the RNase, and the second region comprises a contiguousstretch of non-RNase recruiting nucleoside analogue monomers.

Other “chimeric” oligomers, called “mixmers”, consist of an alternatingcomposition of (i) DNA monomers or nucleoside analogue monomersrecognizable and cleavable by RNase, and (ii) non-RNase recruitingnucleoside analogue monomers.

In some embodiments, in addition to enhancing affinity of the oligomerfor the target region, some nucleoside analogues also mediate RNase(e.g., RNase H) binding and cleavage. Since α-L-LNA monomers recruitRNase activity to a certain extent, in some embodiments, gap regions(e.g., region B as referred to herein below) of oligomers containingα-L-LNA monomers consist of fewer monomers recognizable and cleavable bythe RNase, and more flexibility in the mixmer construction isintroduced.

Gapmer Design

Preferably, the oligomer of the invention is a gapmer.

A “gapmer” is an oligomer which comprises a contiguous stretch ofmonomers capable of recruiting an RNAse, such as RNAseH, such as aregion of at least 6 or 7 DNA monomers, referred to herein as region B,wherein region B is flanked both on its 5′ and 3′ ends by regionsrespectively referred to as regions A and C, each of regions A and Ccomprising or consisting of nucleoside analogues, such asaffinity-enhancing nucleoside analogues, such as 1-6 nucleosideanalogues.

Preferably the gapmer comprises regions, from 5′ to 3′, A-B-C, oroptionally A-B-C-D or D-A-B-C, wherein: region A consists of orcomprises at least one nucleoside analogue, such as at least one LNAmonomer, such as 1-6 nucleoside analogues, such as LNA monomers; andregion B consists of or comprises at least five contiguous monomerswhich are capable of recruiting RNAse (when formed in a duplex with acomplementary target region of the target RNA molecule, such as the mRNAtarget), such as DNA monomers; and region C consists of or comprises atleast one nucleoside analogue, such as at least one LNA monomer, such as1-6 nucleoside analogues, such as LNA monomers, and; region D, whenpresent, consists of or comprises 1, 2 or 3 monomers, such as DNAmonomers.

In various embodiments, region A consists of 1, 2, 3, 4, 5 or 6nucleoside analogues, such as LNA monomers, such as 2-5 nucleosideanalogues, such as 2-5 LNA monomers, such as 3 or 4 nucleosideanalogues, such as 3 or 4 LNA monomers; and/or region C consists of 1,2, 3, 4, 5 or 6 nucleoside analogues, such as LNA monomers, such as 2-5nucleoside analogues, such as 2-LNA monomers, such as 3 or 4 nucleosideanalogues, such as 3 or 4 LNA monomers.

In certain embodiments, region B consists of or comprises 5, 6, 7, 8, 9,10, 11 or 12 contiguous monomers which are capable of recruiting RNAse,or 6-10, or 7-9, such as 8 contiguous monomers which are capable ofrecruiting RNAse. In certain embodiments, region B consists of orcomprises at least one DNA monomer, such as 1-12 DNA monomers,preferably 4-12 DNA monomers, more preferably 6-10 DNA monomers, such as7-10 DNA monomers, most preferably 8, 9 or 10 DNA monomers.

In certain embodiments, region A consists of 3 or 4 nucleosideanalogues, such as LNA monomers, region B consists of 7, 8, 9 or 10 DNAmonomers, and region C consists of 3 or 4 nucleoside analogues, such asLNA monomers. Such designs include (A-B-C) 3-10-3, 3-10-4, 4-10-3,3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3, and mayfurther include region D, which may have one or 2 monomers, such as DNAmonomers.

Further gapmer designs are disclosed in WO 2004/046160, which is herebyincorporated by reference.

U.S. provisional application, 60/977,409, hereby incorporated byreference, refers to “shortmer” gapmer oligomers, which, in certainembodiments, may be the gapmer oligomer according to the invention.

In certain embodiments, the oligomer consists of 10, 11, 12, 13 or 14contiguous monomers, wherein the regions of the oligomer have thepattern (5′-3′), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein;region A consists of 1, 2 or 3 nucleoside analogue monomers, such as LNAmonomers; region B consists of 7, 8 or 9 contiguous monomers which arecapable of recruiting RNAse when formed in a duplex with a complementaryRNA molecule (such as a mRNA target); and region C consists of 1, 2 or 3nucleoside analogue monomers, such as LNA monomers. When present, regionD consists of a single DNA monomer.

In certain embodiments, region A consists of 1 LNA monomer. In certainembodiments, region A consists of 2 LNA monomers. In certainembodiments, region A consists of 3 LNA monomers. In certainembodiments, region C consists of 1 LNA monomer. In certain embodiments,region C consists of 2 LNA monomers. In certain embodiments, region Cconsists of 3 LNA monomers. In certain embodiments, region B consists of7 nucleoside monomers. In certain embodiments, B consists of 8nucleoside monomers. In certain embodiments, region B consists of 9nucleoside monomers. In certain embodiments, region B comprises 1-9 DNAmonomers, such as 2, 3, 4, 5, 6, 7 or 8 DNA monomers. In certainembodiments, region B consists of DNA monomers. In certain embodiments,region B comprises at least one LNA monomer which is in the alpha-Lconfiguration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA monomers in thealpha-L-configuration. In certain embodiments, region B comprises atleast one alpha-L-oxy LNA monomer or wherein all the LNA monomers in thealpha-L-configuration are alpha-L-oxy LNA monomers. In certainembodiments, the number of monomers present in the A-B-C regions of theoligomers is selected from the group consisting of (nucleotide analoguemonomers—region B-nucleoside analogue monomers): 1-8-1, 1-8-2, 2-8-1,2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4, 2-8-4, or; 1-9-1,1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1, 4-9-1, 1-9-4, or;1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, and 3-10-1. In certainembodiments, the number of monomers present in the A-B-C regions of theoligomers of the invention is selected from the group consisting of:2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7-3. In certainembodiments, each of regions A and C consists of two LNA monomers, andregion B consists of 8 or 9 nucleoside monomers, preferably DNAmonomers.

Other gapmer designs include those where regions A and/or C consists of3, 4, 5 or 6 nucleoside analogues, such monomers containing a2′-O-methoxyethyl-ribose sugar (2′MOE) and monomers containing a2′-fluoro-deoxyribose sugar, and region B consists of 8, 9, 10, 11 or 12nucleosides, such as DNA monomers, where regions A-B-C have 5-10-5 or4-12-4 monomers. Further gapmer designs are disclosed in WO2007/146511A2, hereby incorporated by reference.

Linkage Groups

The monomers of the oligomer of the invention are coupled together vialinkage groups. Suitably, each monomer is linked to the 3′ adjacentmonomer via a linkage group.

The terms “linkage group” or “internucleoside linkage” mean a groupcapable of covalently coupling together two contiguous monomers.Specific and preferred examples include phosphate groups (forming aphosphodiester between adjacent nucleoside monomers) andphosphorothioate groups (forming a phosphothioester between adjacentnucleoside monomers).

Suitable linkage groups include those listed within WO 2007/031091, forexample the linkage groups listed on the first paragraph of page 34 ofWO 2007/031091 (hereby incorporated by reference).

It is, in various embodiments, preferred to modify the linkage groupfrom its normal phosphodiester to one that is more resistant to nucleaseattack, such as phosphorothioate or boranophosphate—these two, beingcleavable by RNase H, permitting RNase-mediated antisense inhibition ofexpression of the target gene.

Suitable sulphur (S) containing linkage groups as provided herein may bepreferred. Phosphorothioate linkage groups are also preferred,particularly for the gap region (B) of gapmers. Phosphorothioatelinkages may also be used for the flanking regions (A and C, and forlinking A or C to D, and within region D, as appropriate).

Regions A, B and C, may however comprise linkage groups other thanphosphorothioate, such as phosphodiester linkages, particularly, forinstance when the use of nucleoside analogues protects the linkagegroups within regions A and C from endo-nuclease degradation—such aswhen regions A and C comprise LNA monomers.

The linkage groups in the oligomer may be phosphodiester,phosphorothioate or boranophosphate so as to allow RNase H cleavage ofthe target RNA. Phosphorothioate is preferred, for improved nucleaseresistance and other reasons, such as ease of manufacture.

In various embodiments, adjacent monomers of the oligomer are linked toeach other by means of phosphorothioate groups.

It is recognised that the inclusion of phosphodiester linkages, such asone or two linkages, into an oligomer with a phosphorothioate backbone,particularly with phosphorothioate linkage groups between or adjacent tonucleoside analogue monomers (typically in region A and/or C), canmodify the bioavailability and/or bio-distribution of an oligomer—see WO2008/053314, hereby incorporated by reference.

In some embodiments, such as the embodiments referred to above, wheresuitable and not specifically indicated, all remaining linkage groupsare either phosphodiester or phosphorothioate, or a mixture thereof.

In some embodiments all the internucleoside linkage groups arephosphorothioate.

When referring to specific gapmer oligonucleotide sequences, such asthose provided herein, it will be understood that, in variousembodiments, when the linkages are phosphorothioate linkages,alternative linkages, such as those disclosed herein, may be used, forexample phosphate (phosphodiester) linkages may be used, particularlyfor linkages between nucleoside analogues, such as LNA monomers.Likewise, when referring to specific gapmer oligonucleotide sequences,such as those provided herein, when one or more of the cytosine bases isdenoted as 5-methylcytosine, other cytosine bases present in theoligomer may be unmodified.

Oligomers

In certain embodiments, the oligomers of the invention are selected fromthe group consisting of: SEQ ID NO 133-152, as shown in Table 2.

In Table 2, upper case letters indicate nucleoside analogue monomers andsubscript “s” represents a phosphorothiate linkage. Lower case lettersrepresent nucleoside (DNA) monomers. Absence of “s” (if any) indicates aphosphodiester linkage.

TABLE 2 Oligomer designs Length SEQ ID NO Sequence (5′-3′) (bases)SEQ ID NO: 133 G s A s Asa_(s)g_(s)c_(s)t_(s)g_(s)a_(s)t_(s)g_(s)g_(s)a_(s) C s C s A 16SEQ ID NO: 134 C s A s Gsa_(s)c_(s)t_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)t_(s) G s G s C 16SEQ ID NO: 135 C s A s Gsa_(s)a_(s)t_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)g_(s) T s G s A 16SEQ ID NO: 136 G s C s Asc_(s)t_(s)g_(s)c_(s)c_(s)a_(s)t_(s)t_(s)t_(s)t_(s) A s G s C 16SEQ ID NO: 137 G s T s Asa_(s)t_(s)a_(s)g_(s)c_(s)c_(s)a_(s)a_(s)g_(s)a_(s) A s T s T 16SEQ ID NO: 138 A s C s Tsc_(s)t_(s)g_(s)c_(s)t_(s)t_(s)g_(s)t_(s)g_(s)g_(s) T s C s C 16SEQ ID NO: 139 C s C s Asc_(s)c_(s)a_(s)g_(s)c_(s)t_(s)t_(s)c_(s)t_(s)a_(s) C s C s A 16SEQ ID NO: 140 G s A s Gst_(s)c_(s)c_(s)a_(s)a_(s)a_(s)g_(s)a_(s)c_(s)a_(s) G s T s T 16SEQ ID NO: 141 A s C s Csc_(s)a_(s)c_(s)t_(s)t_(s)g_(s)g_(s)c_(s)a_(s)g_(s) A s C s C 16SEQ ID NO: 142 G s C s Asc_(s)a_(s)a_(s)a_(s)c_(s)a_(s)ast_(s)g_(s)g_(s) A s A s T 16SEQ ID NO: 143 G s C s Asg_(s)c_(s)t_(s)a_(s)c_(s)t_(s)c_(s)t_(s)t_(s)t_(s) G s G s A 16SEQ ID NO: 144 C s T s Csc_(s)c_(s)t_(s)c_(s)a_(s)g_(s)c_(s)t_(s)t_(s)c_(s) A s A s T 16SEQ ID NO: 145 G s C s Asg_(s)t_(s)c_(s)t_(s)c_(s)a_(s)t_(s)t_(s)c_(s)c_(s) A s A s G 16SEQ ID NO: 146 T s A s Tsc_(s)c_(s)a_(s)c_(s)c_(s)a_(s)g_(s)a_(s)g_(s)t_(s) G s A s A 16SEQ ID NO: 147 C s A s Tsc_(s)c_(s)a_(s)t_(s)g_(s)a_(s)g_(s)g_(s)t_(s)c_(s) C s T s G 16SEQ ID NO: 148 C s C s Ast_(s)c_(s)t_(s)t_(s)g_(s)t_(s)g_(s)a_(s)t_(s)c_(s) C s A s T 16SEQ ID NO: 149 A s A s Gsc_(s)a_(s)a_(s)g_(s)c_(s)a_(s)a_(s)a_(s)g_(s)t_(s) C s A s G 16SEQ ID NO: 150 G s A s Asa_(s)t_(s)t_(s)g_(s)c_(s)t_(s)g_(s)t_(s)a_(s)g_(s) C s A s G 16SEQ ID NO: 151 G s T s Gst_(s)t_(s)c_(s)t_(s)a_(s)c_(s)a_(s)csc_(s)a_(s) T s T s A 16SEQ ID NO: 152 A s A s Csa_(s)t_(s)g_(s)a_(s)a_(s)a_(s)t_(s)a_(s)g_(s)a_(s) T s C s C 16

Conjugates

In the context of this disclosure, the term “conjugate” indicates acompound formed by the covalent attachment (“conjugation”) of anoligomer, as described herein, to one or more moieties that are notthemselves nucleic acids or monomers (“conjugated moiety”). Examples ofsuch conjugated moieties include macromolecular compounds such asproteins, fatty acid chains, sugar residues, glycoproteins, polymers, orcombinations thereof. Typically, proteins may be antibodies for a targetprotein. Typical polymers may be polyethylene glycol. WO 2007/031091provides suitable moieties and conjugates, which are hereby incorporatedby reference.

Accordingly, provided herein are conjugates comprising an oligomer asherein described, and at least one conjugated moiety that is not anucleic acid or monomer, covalently attached to said oligomer.Therefore, in certain embodiments, where the oligomer of the inventionconsists of contiguous monomers having a specified sequence of bases, asherein disclosed, the conjugate may also comprise at least oneconjugated moiety that is covalently attached to said oligomer.

In certain embodiments, the oligomer is conjugated to a moiety thatincreases the cellular uptake of oligomeric compounds.

Conjugates may enhance the activity, cellular distribution or cellularuptake of the oligomer of the invention. Such moieties include, but arenot limited to, antibodies, polypeptides, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g. Hexyl-s-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipids, e.g., dihexadecyl-rac-glycerol ortriethylammonium 1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, apolyamine or a polyethylene glycol chain, an adamantane acetic acid, apalmityl moiety, an octadecylamine or hexylamino-carbonyl-oxycholesterolmoiety.

The oligomers of the invention may also be conjugated to active drugsubstances, for example, aspirin, ibuprofen, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic.

In certain embodiments, the conjugated moiety is a sterol, such ascholesterol.

In various embodiments, the conjugated moiety comprises or consists of apositively charged polymer, such as a positively charged peptide of, forexample 1-50, such as 2-20 such as 3-10 amino acid residues in length,and/or polyalkylene oxide such as polyethylglycol(PEG) or polypropyleneglycol—see WO 2008/034123, hereby incorporated by reference. Suitably,the positively charged polymer, such as a polyalkylene oxide may beattached to the oligomer of the invention via a linker such as thereleasable linker described in WO 2008/034123.

Activated Oligomers

The term “activated oligomer,” as used herein, refers to an oligomer ofthe invention that is covalently linked (i.e., functionalized) to atleast one functional moiety that permits covalent linkage of theoligomer to one or more conjugated moieties, i.e., moieties that are notthemselves nucleic acids or monomers, to form the conjugates hereindescribed. Typically, a functional moiety will comprise a chemical groupthat is capable of covalently bonding to the oligomer via, e.g., a3′-hydroxyl group or the exocyclic NH2 group of the adenine base, aspacer that in some embodiments is hydrophilic and a terminal group thatis capable of bill ding to a conjugated moiety (e.g., an amino,sulfhydryl or hydroxyl group). In some embodiments, this terminal groupis not protected, e.g., is an NH2 group. In other embodiments, theterminal group is protected, for example, by any suitable protectinggroup such as those described in “Protective Groups in OrganicSynthesis” by Theodora W Greene and Peter G M Wuts, 3rd edition (JohnWiley & Sons, 1999). Examples of suitable hydroxyl protecting groupsinclude esters such as acetate ester, aralkyl groups such as benzyl,diphenylmethyl, or triphenylmethyl, and tetrahydropyrallY1. Examples ofsuitable amino protecting groups include benzyl, alpha-methylbenzyl,diphenylmethyl, triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl,and acyl groups such as trichloroacetyl or trifluoroacetyl.

In some embodiments, the functional moiety is self-cleaving. In otherembodiments, the functional moiety is biodegradable. See e.g., U.S. Pat.No. 7,087,229, which is incorporated by reference herein in itsentirety.

In some embodiments, oligomers of the invention are functionalized atthe 5′ end in order to allow covalent attachment of the conjugatedmoiety to the 5′ end of the oligomer. In other embodiments, oligomers ofthe invention can be functionalized at the 3′ end. In still otherembodiments, oligomers of the invention can be functionalized along thebackbone or on the heterocyclic base moiety. In yet other embodiments,oligomers of the invention can be functionalized at more than oneposition independently selected from the 5′ end, the 3′ end, thebackbone and the base.

In some embodiments, activated oligomers of the invention aresynthesized by incorporating during the synthesis one or more monomersthat is covalently attached to a functional moiety. In otherembodiments, activated oligomers of the invention are synthesized withmonomers that have not been functionalized, and the oligomer isfunctionalized upon completion of synthesis.

In some embodiments, the oligomers are functionalized with a hinderedester containing an aminoalkyl linker, wherein the alkyl portion has theformula (CH2)w, wherein w is an integer ranging from 1 to 10, preferablyabout 6, wherein the alkyl portion of the alkylamino group can bestraight chain or branched chain, and wherein the functional group isattached to the oligomer via an ester group (—O—C(O)—(CH2)wNH).

In other embodiments, the oligomers are functionalized with a hinderedester containing a (CH2)w-sulfhydryl (SH) linker, wherein w is aninteger ranging from 1 to 10, preferably about 6, wherein the alkylportion of the alkylamino group can be straight chain or branched chain,and wherein the functional group attached to the oligomer via an estergroup (—O—C(O)—(CH2)wSH). In some embodiments, sulfhydryl-activatedoligonucleotides are conjugated with polymer moieties such aspolyethylene glycol or peptides (via formation of a disulfide bond).

Activated oligomers covalently linked to at least one functional moietycan be synthesized by any method known in the art, and in particular bymethods disclosed in U.S. Patent Publication No. 2004/0235773, which isincorporated herein by reference in its entirety, and in Zhao et al.(2007) J. Controlled Release 119: 143-152; and Zhao et al. (2005)Bioconjugate Chem. 16:758-766.

In still other embodiments, the oligomers of the invention arefunctionalized by introducing sulfhydryl, amino or hydroxyl groups intothe oligomer by means of a functionalizing reagent substantially asdescribed in U.S. Pat. Nos. 4,962,029 and 4,914,210, i.e., asubstantially linear reagent having a phosphoramidite at one end linkedthrough a hydrophilic spacer chain to the opposing end which comprises aprotected or unprotected sulfhydryl, amino or hydroxyl group. Suchreagents primarily react with hydroxyl groups of the oligomer. In someembodiments, such activated oligomers have a functionalizing reagentcoupled to a 5′-hydroxyl group of the oligomer. In other embodiments,the activated oligomers have a functionalizing reagent coupled to a3′-hydroxyl group. In still other embodiments, the activated oligomersof the invention have a functionalizing reagent coupled to a hydroxylgroup on the backbone of the oligomer. In yet further embodiments, theoligomer of the invention is functionalized with more than one of thefunctionalizing reagents as described in U.S. Pat. Nos. 4,962,029 and4,914,210, incorporated herein by reference in their entirety. Methodsof synthesizing such functionalizing reagents and incorporating theminto monomers or oligomers are disclosed in U.S. Pat. Nos. 4,962,029 and4,914,210.

In some embodiments, the 5′-terminus of a solid-phase bound oligomer isfunctionalized with a dienyl phosphoramidite derivative, followed byconjugation of the deprotected oligomer with, e.g., an amino acid orpeptide via a Diels-Alder cycloaddition reaction.

In various embodiments, the incorporation of monomers containing2′-sugar modifications, such as a 2′-carbamate substituted sugar or a2′-(O-pentyl-N-phthalimido)deoxyribose sugar into the oligomerfacilitates covalent attachment of conjugated moieties to the sugars ofthe oligomer. In other embodiments, an oligomer with an amino-containinglinker at the 2′-position of one or more monomers is prepared using areagent such as, for example, 5′dimethoxytrityl-2′-O-(e-phthalimidylaminopentyl)-2′-deoxyadenosine-3′-N,N-diisopropyl-cyanoethoxyphosphoramidite. See. e.g., Manoharan, et al., Tetrahedron Letters,1991, 34, 7171.

In still further embodiments, the oligomers of the invention haveamine-containing functional moieties on the nucleobase, including on theN6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or5 positions of cytosine. In some embodiments, such functionalization maybe achieved by using a commercial reagent that is already functionalizedin the oligomer synthesis.

Some functional moieties are commercially available, for example,heterobifunctional and homobifunctional linking moieties are availablefrom the Pierce Co. (Rockford, Ill.). Other commercially availablelinking groups are 5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents,both available from Glen Research Corporation (Sterling, Va.).5′-Amino-Modifier C6 is also available from ABI (Applied BiosystemsInc., Foster City, Calif.) as Aminolink:-2, and 3′Amino-Modifier is alsoavailable from Clontech Laboratories Inc. (Palo Alto, Calif.).

Compositions

The oligomer of the invention may be used in pharmaceutical formulationsand compositions. Suitably, such compositions comprise apharmaceutically acceptable diluent, carrier, salt or adjuvant. WO2007/031091 provides suitable and preferred pharmaceutically acceptablediluents, carriers and adjuvants—which are hereby incorporated byreference. Suitable dosages, formulations, administration routes,compositions, dosage forms, combinations with other therapeutic agents,pro-drug formulations arc also provided in WO 2007/031091—which are alsohereby incorporated by reference.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the desired biological activity of the hereinidentified compounds and exhibit acceptable levels of undesired toxiceffects. Non-limiting examples of such salts can be formed with organicamino acid and base addition salts formed with metal cations such aszinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt,nickel, cadmium, sodium, potassium, and the like, or with a cationformed from ammonia, N,N-dibenzylethylene-diamine, D-glucosamine,tetraethylammonium, or ethylenediamine; or (c) combinations of (a) and(b); e.g., a zinc tannate salt or the like.

Applications

The term “treatment” as used herein refers to both treatment of anexisting disease (e.g. a disease or disorder as referred to hereinbelow), or prevention of a disease, i.e. prophylaxis. It will thereforebe recognised that, in certain embodiments, “treatment” includesprophylaxis.

The oligomers of the invention may be utilized as research reagents for,for example, diagnostics, therapeutics and prophylaxis.

In research, such oligomers may be used to specifically inhibit theexpression of beta-catenin protein (typically, by degrading orinhibiting beta-catenin mRNA and thereby preventing protein formation)in cells and experimental animals, thereby facilitating functionalanalysis of the target or an appraisal of its usefulness as a target fortherapeutic intervention.

In diagnostics, the oligomers may be used to detect and quantitatebeta-catenin expression in cells and tissues by northern blotting,in-situ hybridisation or similar techniques.

For therapeutics, a non-human animal or a human, suspected of having adisease or disorder which can be treated by modulating the expression ofbeta-catenin, is treated by administering an effective amount of anoligomer (or conjugate thereof) in accordance with this invention.Further provided are methods of treating a mammal, such as treating ahuman, suspected of having or being prone to a disease or condition,associated with expression of beta-catenin, by administering atherapeutically or prophylactically effective amount of one or more ofthe oligomers or compositions of the invention.

The invention also provides for the use of the compounds or conjugatesof the invention as described for the manufacture of a medicament forthe treatment of a disorder as referred to herein, or for a method ofthe treatment of a disorder as referred to herein.

The invention also relates to an oligomer, a composition or a conjugateas described herein for use as a medicament.

The invention also provides for a method for treating a disorder asreferred to herein, the method comprising administering an effectiveamount of a compound according to the invention as herein described,and/or an effective amount of a conjugate according to the invention,and/or a pharmaceutical composition according to the invention to apatient in need thereof.

In various embodiments, the oligomer, or conjugate thereof, induces adesired therapeutic effect in humans through, for example, hydrogenbonding to a target nucleic acid. The oligomer causes a decrease(inhibition) in the expression of a target via hydrogen bonding(hybridisation) to the mRNA of the target thereby resulting in areduction in gene expression. It is also envisaged that the oligomersand conjugates disclosed herein may have non-therapeutic applications,such as diagnostic applications.

It is highly preferred that the compounds of the invention are capableof hybridising to the target nucleic acid, such as beta-catenin mRNA, byWatson-Crick base pairing.

Medical Indications

In certain therapeutic embodiments, the disorder to be treated isselected from the group consisting of a hyperproliferative disorder,such as cancer, such as a cancer selected from the group consisting ofcolorectal cancer, hepatocellular cancer, endometrial cancer, malignantmelanoma, ovarian cancer, pancreatic cancer, pituitary cancer,oesophageal cancer, lung cancer, breast cancer, kidney cancer,haematopoetic system cancer, cervical cancer, CNS cancer, bone cancer,biliary tract cancer and adrenal gland cancer

In certain embodiments, the disorder, is a cancer selected from thegroup consisting of colorectal cancer, hepatocellular cancer,endometrial cancer, and malignant melanoma.

In certain embodiments, the disorder is a cancer selected from the groupconsisting of liver cancer and kidney cancer.

In certain embodiments, the disease or disorder is associated with amutation in the beta-catenin gene or a gene whose protein product isassociated with or interacts with beta-catenin, such as the APC gene.Therefore, in various embodiments, the target mRNA is a mutated form ofthe beta-catenin sequence, for example it may comprise one or moresingle point mutations, such as SNPs associated with cancer.

Examples of such diseases where mutations in the beta-catenin or APCgene lead to abnormal levels of beta-catenin activity are: (1)Colorectal cancer, APC and beta-catenin are mutually mutated in morethan 70% of all cases (Powell et al., Nature, 1992; Morin et al.,Science, 1997; Sparks et al., Cancer Res, 1998); (2) Hepatocellularcancer, beta-catenin are mutated in more than 25% of cases (de La CosteA, PNAS, 1998); (3) Endometrial cancer, beta-catenin are mutated>10%;and (4) Malignant melanoma, Beta-catenin are mutated >10% (Rubinfeld etat., Science, 1997).

Further examples of such diseases are cancer of the ovary, pancreas,pituitary, oesophagus, lung, breast, kidney, haematopoetic system,cervix, CNS, bone, biliary tract and adrenal gland. It has been shownthat mutations in the beta-catenin or APC gene are associated with thesediseases (Catalogue of Somatic Mutations in Cancer available from theSanger Institute (United Kingdom)).

In certain embodiments, the disease or disorder is associated withabnormal levels of a mutated form of beta-catenin. In certainembodiments, the disease or disorder is associated with abnormal levelsof a wild-type form of beta-catenin. One aspect of the invention isdirected to a method of treating a mammal suffering from or susceptibleto conditions associated with abnormal levels of beta-catenin,comprising administering to the mammal a therapeutically effectiveamount of an oligomer of the invention targeted to beta-catenin orvarious compositions or conjugates thereof. In some embodiments, theoligomer comprises one or more LNA monomers.

Suitable dosages, formulations, administration routes, compositions,dosage forms, combinations with other therapeutic agents, pro-drugformulations are also provided in WO 2007/031091—which are herebyincorporated by reference. The invention also provides for apharmaceutical composition comprising a compound or a conjugate asherein described or a conjugate, and a pharmaceutically acceptablediluent, carrier or adjuvant. WO 2007/031091 provides suitable andpreferred pharmaceutically acceptable diluent, carrier andadjuvants—which are hereby incorporated by reference.

Furthermore, the invention described herein encompasses a method ofpreventing or treating a disease comprising administering atherapeutically effective amount of a beta-catenin modulating oligomerto a human in need of such therapy. The invention further encompassesthe use of a short period of administration of a beta-catenin modulatingoligonucleotide compound (oligomer or conjugate).

In various embodiments, the invention is drawn to a method of treatingabnormal levels of beta-catenin comprising administering (i) aneffective amount of a conjugate of the invention, or compositionsthereof, and (ii) an effective amount of a second agent. In someembodiments, administration of the conjugate and the second agent issimultaneous. In other embodiments, administration of the conjugate andthe second agent is sequential. Typically, the second agent iscovalently linked to the oligomer to form the conjugate.

Further Embodiments

The following are further embodiments, and may be combined with theembodiments

7. EXAMPLES Example 1 Monomer Synthesis

The LNA monomer building blocks and derivatives were prepared followingpublished procedures and references cited therein—see WO 07/031,081 andthe references cited therein.

Example 2 Oligonucleotide Synthesis

Oligonucleotides were synthesized according to the method described inWO 07/031,081. Table 1 shows examples of antisense oligonucleotidemotifs and of the invention.

Example 3 Design of the Oligonucleotides

In accordance with the invention, a series of oligonucleotides weredesigned to target different regions of the human beta-catenin mRNAusing the published sequence, GenBank accession number NM_(—)001904,presented herein as SEQ ID NO: 173.

Table 2 shows oligomer designs of the invention. Table 3 shows 24mersequence motifs from which oligomers of the invention may bedesigned—the bold type represents 16mer sequence motifs as shown inTable 1 that are incorporated into the longer oligomers.

TABLE 3 Beta-Catenin 24mers Sequences Short-mer Compound 24-mer16mer SEQ IDS SEQ IDS ID 24-mer SEQ IDS SEQ ID SEQ ID NO: 1  2-15133-153 ttta gaaagctgatggacca taac 174 SEQ ID NO: 16 134-154 cctccagacttaaagatggc cagt 175 SEQ ID NO: 17 135-155 aaca cagaatccactggtgaacca 176 SEQ ID NO: 18 19-32 136-156 aaac gcactgccattttagc tcct 177SEQ ID NO: 33 137-157 tgtc gtaatagccaagaatt taac 178 SEQ ID NO: 34 35-48138-158 cagc actctgcttgtggtcc acag 179 SEQ ID NO: 49 139-159 cattccaccagcttctacaa tagc 180 SEQ ID NO: 50 140-160 ctga gagtccaaagacagttctga 181 SEQ ID NO: 51 141-161 tacc acccacttggcagacc atca 182SEQ ID NO: 52 142-162 agct gcacaaacaatggaat ggta 183 SEQ ID NO: 53143-163 ccct gcagctactctttgga tgtt 184 SEQ ID NO: 54 144-164 gtggctccctcagcttcaat agct 185 SEQ ID NO: 55 145-165 atca gcagtctcattccaagccat 186 SEQ ID NO: 56 146-166 gcca tatccaccagagtgaa aaga 187SEQ ID NO: 57 147-167 agcc catccatgaggtcctg ggca 188 SEQ ID NO: 58 59-72148-168 aatt ccatcttgtgatccat tctt 189 SEQ ID NO: 73 74-87 149-169 ttcaaagcaagcaaagtcag tacc 190 SEQ ID NO: 88 89-102 150-170 attagaaattgctgtagcag tatt 191 SEQ ID NO: 103  104-117 151-171 attagtgttctacaccatta ctca 192 SEQ ID NO: 118  119-132 152-172 caaaaacatgaaatagatcc acct 193

Example 4 In Vitro Model Cell Culture

The effect of antisense oligonucleotides on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. Target nucleicacids can be expressed endogenously or by transient or stabletransfection. The expression level of the target nucleic acid can beroutinely determined using, for example, Northern blot analysis,Real-Time PCR, Ribonuclease protection assays. The following cell typesare provided for illustrative purposes, but other cell types can beroutinely used, provided that the target is expressed in the cell typechosen.

Cells were cultured in the appropriate medium as described below andmaintained at 37° C. at 95-98% humidity and 5% C02. Cells were routinelypassaged 2-3 times weekly.

SW480: The human colorectal cancer cell line SW480 was cultured in L-15medium (Leibovitz)+10% fetal bovine serum (FBS)+Glutamax I+pen/strep.

HCT116: The human colorectal cancer cell line HCT116 was cultured inMcCoy's 5a modified medium (Sigma)+10% fetal bovine serum (FBS)+GlutamaxI+pen/strep.

Example 5 In Vitro Model Treatment with Antisense Oligonucleotide

The cells were treated with oligonucleotide using the cationic liposomeformulation LipofectAMINE 2000 (Gibco) as transfection vehicle. Cellswere seeded in 6-well cell culture plates (NUNC) and treated when 75-90%confluent. Oligonucleotide concentrations used ranged from 1 nM to 25 nMfinal concentration. Formulation of oligonucleotide-lipid complexes werecarried out essentially as described by the manufacturer usingserum-free OptiMEM (Gibco) and a final lipid concentration of 10 μg/mLLipofectAMINE 2000. Cells were incubated at 37° C. for 4 hours andtreatment was stopped by removal of oligonucleotide-containing culturemedium. Cells were washed and serum-containing media was added. Afteroligonucleotide treatment cells were allowed to recover for 20 hoursbefore they were harvested for RNA analysis.

Example 6 In Vitro Model Extraction of RNA and cDNA Synthesis

For RNA isolation from the cell lines, the RNeasy mini kit (Qiagen cat.no. 74104) was used according to the protocol provided by themanufacturer.

First strand synthesis was performed using Reverse Transcriptasereagents from Ambion according to the manufacturer's instructions.

For each sample 0.5 μg total RNA was adjusted to 10.8 μl with RNase freeH20 and mixed with 2 μl random decamers (50 μM) and 4 μl dNTP mix (2.5mM each dNTP) and heated to 70° C. for 3 min after which the sampleswere rapidly cooled on ice. After cooling the samples on ice, 2 μl 10×Buffer RT, 1 μl MMLV Reverse Transcriptase (100 U/μl) and 0.25 μl RNaseinhibitor (10 U/μl) was added to each sample, followed by incubation at42° C. for 60 min, heat inactivation of the enzyme at 95° C. for 10 minand then the sample was cooled to 4° C.

Example 7 In Vitro Model Analysis of Oligonucleotide Inhibition ofBeta-Catenin Expression by Real-Time PCR

Antisense modulation of beta-catenin expression can be assayed in avariety of ways known in the art. For example, beta-catenin mRNA levelscan be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or real-time PCR. Real-timequantitative PCR is presently preferred. RNA analysis can be performedon total cellular RNA or mRNA. Methods of RNA isolation and RNA analysissuch as Northern blot analysis is routine in the art and is taught in,for example, Current Protocols in Molecular Biology, John Wiley andSons.

Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available Multi-Color Real Time PCR Detection System,available from Applied Biosystem.

7.2.1 Real-time Quantitative PCR Analysis of Betacatenin mRNA Levels

The sample content of human beta-catenin mRNA was quantified using thehuman beta-catenin ABI Prism Pre-Developed TaqMan Assay Reagent (AppliedBiosystems cat. no. Hs00170025_ml) according to the manufacturer'sinstructions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA quantity was usedas an endogenous control for normalizing any variance in samplepreparation.

The sample content of human GAPDH mRNA was quantified using the humanGAPDH ABI Prism Pre-Developed TaqMan Assay Reagent (Applied Biosystemscat. no. 4310884E) according to the manufacturer's instructions.

Real-time Quantitative PCR is a technique well known in the art and istaught in for example Heid et al. Real time quantitative PCR, GenomeResearch (1996), 6: 986-994.

7.2.2 Real Time PCR

The cDNA from the first strand synthesis performed as described inExample 8 was diluted 2-20 times, and analyzed by real time quantitativePCR using Taqman 7500 FAST from Applied Biosystems. The primers andprobe were mixed with 2× Taqman Fast Universal PCR master mix (2×)(Applied Biosystems Cat.# 4364103) and added to 4/μl cDNA to a finalvolume of 10/μl. Each sample was analysed in triplicate. Assaying 2 folddilutions of a cDNA that had been prepared on material purified from acell line expressing the RNA of interest generated standard curves forthe assays. Sterile H20 was used instead of cDNA for the no templatecontrol. PCR program: 95° C. for 30 seconds, followed by 40 cycles of95° C., 3 seconds, 60° C., 30 seconds. Relative quantities of targetmRNA sequence were determined from the calculated Threshold cycle usingthe Applied Biosystems Fast System SDS Software Version 1.3.1.21.

7.2.3 Example 8 In Vitro Analysis Antisense Inhibition of HumanBeta-Catenin Expression by Oligonucleotides

Oligonucleotides from Table 2 were evaluated for their potential toknockdown beta-catenin mRNA at concentrations of 1, 5, and 25 nM inSW480 cells (see FIG. 2). Results are tabulated in Table 4, in which thedata are presented as percentage down-regulation of beta-catenin mRNArelative to mock transfected cells at 5 nM. Lower case letters representDNA monomers, bold upper case letters represent β-D-oxy-LNA monomers.The cytosine bases of all LNA monomers are 5′ methyl cytosines.Subscript “s” represents phosphorothiate linkage.

TABLE 4  Beta- catenin Test substance Sequence (5′-3′) (% inhib.)SEQ ID NO: 153 G s A s Asa_(s)g_(s)c_(s)t_(s)g_(s)a_(s)t_(s)g_(s)g_(s)a_(s) C s 88.11% C s ASEQ ID NO: 154 C s A s Gsa_(s)c_(s)t_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)t_(s) G s 86.4% G s CSEQ ID NO: 155 C s A s Gsa_(s)a_(s)t_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)g_(s) T s 76.6% G s ASEQ ID NO: 156 G s C s Asc_(s)t_(s)g_(s)c_(s)c_(s)a_(s)t_(s)t_(s)t_(s)t_(s) A s 89.2% G s CSEQ ID NO: 157 G s T s Asa_(s)t_(s)a_(s)g_(s)c_(s)c_(s)a_(s)a_(s)g_(s)a_(s) A s 56.4% T s TSEQ ID NO: 158 A s C s Tsc_(s)t_(s)g_(s)c_(s)t_(s)t_(s)g_(s)t_(s)g_(s)g_(s) T s 77.6% C s CSEQ ID NO: 159 C s C s Asc_(s)c_(s)a_(s)g_(s)c_(s)t_(s)t_(s)c_(s)t_(s)a_(s) C s 49.7% A s ASEQ ID NO: 160 G s A s Gst_(s)c_(s)c_(s)a_(s)a_(s)a_(s)g_(s)a_(s)c_(s)a_(s) G s 60.1% T s TSEQ ID NO: 161 A s C s Csc_(s)a_(s)c_(s)t_(s)t_(s)g_(s)g_(s)c_(s)a_(s)g_(s) A s 75.6% C s CSEQ ID NO: 162 G s C s Asc_(s)a_(s)a_(s)a_(s)c_(s)a_(s)a_(s)t_(s)g_(s)g_(s) A s 10.8% A s TSEQ ID NO: 163 G s C s Asg_(s)c_(s)t_(s)a_(s)c_(s)t_(s)c_(s)t_(s)t_(s)t_(s) G s 48.3% G s ASEQ ID NO: 164 C s T s Csc_(s)c_(s)t_(s)c_(s)a_(s)g_(s)c_(s)t_(s)t_(s)c_(s) A s 35.5% A s TSEQ ID NO: 165 G s C s Asg_(s)t_(s)c_(s)t_(s)c_(s)a_(s)t_(s)t_(s)c_(s)c_(s) A s 25.5% A s GSEQ ID NO: 166 T s A s Tsc_(s)c_(s)a_(s)c_(s)c_(s)a_(s)g_(s)a_(s)g_(s)t_(s) G s 27.3% A s ASEQ ID NO: 167 C s A s Tsc_(s)c_(s)a_(s)t_(s)g_(s)a_(s)g_(s)g_(s)t_(s)c_(s) C s 24.5% T s GSEQ ID NO: 168 C s C s Ast_(s)c_(s)t_(s)t_(s)g_(s)t_(s)g_(s)a_(s)t_(s)c_(s) C s 74.4% A s TSEQ ID NO: 169 A s A s Gsc_(s)a_(s)a_(s)g_(s)c_(s)a_(s)a_(s)a_(s)g_(s)t_(s) C s 87.5% A s GSEQ ID NO: 170 G s A s Asa_(s)t_(s)t_(s)g_(s)c_(s)t_(s)g_(s)t_(s)a_(s)g_(s) C s 91.5% A s GSEQ ID NO: 171 G s T s Gst_(s)t_(s)c_(s)t_(s)a_(s)c_(s)a_(s)c_(s)c_(s)a_(s) T s 94.9% T s ASEQ ID NO: 172 A s A s Csa_(s)t_(s)g_(s)a_(s)a_(s)a_(s)t_(s)a_(s)g_(s)a_(s) T s 93.6% C s CSEQ ID NO: 194 C s G s T s c s a s g s t s a s t s g s c s g s A s A s Ts c

As shown in Table 4, oligonucleotides of SEQ ID NOs: 153, 154, 155, 156,158, 161, 168, 169, 170, 171 and 172 demonstrated about 74% or greaterinhibition of beta-catenin mRNA expression at 5 nM in these experimentsand are therefore preferred.

Also preferred are oligonucleotides based on the illustrated antisenseoligomer sequences, for example varying the length (shorter or longer)and/or monomer content (e.g. the type and/or proportion of nucleosideanalogue monomers), which also provide good inhibition of beta-cateninexpression.

7.2.4 Example 9 In Vitro Analysis Western Blot Analysis of Beta-CateninProtein Levels

The in vitro effect of oligomeric compounds on beta-catenin proteinlevels in transfected cells was determined by Western Blotting. 200.000SW480 cells transfected with oligonucleotides as described in Example 5were harvested and lysed in RIPA lysis buffer (50 mM Tris-HCl pH7.4, 150mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium Deoxycholate)supplemented with protease inhibitor cocktail (Roche). Total proteinconcentrations were measured using a BCA protein assay kit (Pierce). 50μg total protein was run on 3-8% Tris Acetate gels and blotted onto PVDFmembranes according to manufacturer's instructions (Invitrogen). Afterovernight incubation in blocking buffer (PBS•-T supplemented with 5% lowfat milk powder), the membranes were incubated overnight with ananti-beta-catenin antibody (BD Transduction laboratories). As control ofloading tubulin was detected using monoclonal antibodies from Neomarker.Membranes were then incubated with secondary antibodies and Beta-cateninor tubluin proteins were visualized using a chemiluminescenceECL+detection kit (Amersham). See FIG. 3.

7.2.5 Example 10 Measurement of Proliferating Viable Cells (MTS Assay)

HCT116 colorectal cancer cells were seeded to a density of 200,000 cellsper well in a 6 well plate in McCoy's 5a modified medium (Sigma M8403)+2mM Glutamax I (Gibco 35050-(38)+10% FBS (Brochrom #193575010)+25 μg/μlGentamicin (Sigma G 1397 50 mg/ml) the day prior to transfection. Thenext day medium was removed followed by addition of 1.2 ml OptiMEMcontaining 7.5 μg/ml Lipofectamine-2000 (Invitrogen). Cells wereincubated for 7 min before adding 0.3 ml oligonucleotides diluted inOptiMEM. The final oligonucleotide concentration was 25 nM. After 4hours of treatment, media was removed and cells were trypsinized andseeded to a density of 5000 cells per well in clear 96 well plates(Scientific Orange no. 1472030100) in 100 μl McCoy's 5a modified medium(Sigma M8403)+2 mM Ghltamax I (Gibco 35050-038)+10% FBS (Brochrom#193575010)+25 μgilll Gentamicin (Sigma G1397 50 mg/ml). Viable cellswere measured at the times indicated by adding 10 μl the tetrazoliumcompound[3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and an electron coupling reagent (phenazineethosulfate; PES) (CellTiter 96® AQueous One Solution Cell ProliferationAssay, Promega). Viable cells were measured at 490 nm in a Powerwave(Biotek Instruments). The OD490 nm were plotted against time/h. (SeeFIG. 4).

7.2.6 Example 11 Inhibition of Beta-Catenin mRNA in Mouse Liver

NMRI mice were dosed i.v. with 25 mg/kg oligonucleotide (SEQ ID NO: 156,158, 168, 169, 171) on three consecutive days (group size of 5 mice).The antisense oligonucleotides were dissolved in 0.9% saline (NaCl).Animals were sacrificed 24 h after last dosing and liver tissue wassampled and stored in RNA later until RNA extraction and QPCR analysis.Total RNA was extracted and beta-catenin mRNA expression in liversamples was measured by QPCR as described in Example 7 using a mousebeta-catenin QPCR assay (cat. Mm00483033_ml, Applied Biosystems).Results were normalised to mouse GAPDH (cat. no. 4352339E, AppliedBiosystems) and plotted relative to saline treated controls, see FIG. 5.

7.2.7 Example 12 Preparation of a Conjugate of SEQ In NO: 161 andPolyethylene Glycol

The oligomers having SEQ ID NO: 161, SEQ ID NO: 168 and SEQ ID NO: 171are functionalized on the 5′ terminus by attaching an amino alkyl group,such as hexan-1-amine blocked with a blocking group such as Fmoc to the5′ phosphate groups of the oligomers using routine phosphoramiditechemistry, oxidizing the resultant compounds, deprotecting them andpurifying them to achieve the functionalized oligomers, respectively,having the formulas (IA), (IB) and (IC):

wherein the bold capital letters and the subscript “s” in SEQ ID NOs:161, 168 and 171 have the same meaning as discussed above, and MeC is5-methylcytosine.

A solution of activated PEG, such as the one shown in formula (II):

wherein the PEG moiety has an average molecular weight of 12,000, andeach of the compounds of formulas (IA), (IB) and (C) in PBS buffer arestirred in separate vessels at room temperature for 12 hours. Thereaction solutions are extracted three times with methylene chloride andthe combined organic layers arc dried over magnesium sulphate andfiltered and the solvent is evaporated under reduced pressure. Theresulting residues are dissolved in double distilled water and loadedonto an anion exchange column. Unreacted PEG linker is eluted with waterand the products are eluted with NH4HCO3 solution. Fractions containingpure products are pooled and lypophilized to yield the conjugates SEQ IDNOs: 161, 168 and 171, respectively as show in formulas (IIIA), (IIIB)and (IIIC):

wherein each of the oligomers of SEQ ID NOs: 161, 168 and 171 isattached to a PEG polymer having average molecular weight of 12,000 viaa linker.

8. SPECIFIC EMBODIMENTS, CITATION OF REFERENCES

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various references, including patent applications, patents, andscientific publications, are cited herein; the disclosure of each suchreference is hereby incorporated herein by reference in its entirety.

1. An antisense oligonucleotide (such as the oligomer) capable ofbinding to a target sequence of the beta-catenin gene of SEQ ID NO: 173or allele thereof and down-regulating expression of beta-catenin, saidoligonucleotide comprising a sequence of 10-50 nucleobases correspondingto the target sequence.
 2. The antisense oligonucleotide of embodiment1, wherein the target sequence is selected from regions of thebeta-catenin gene represented by SEQ ID NOS: 1-132 or an allelic variantthereof.
 3. The oligonucleotide of embodiment 1 or embodiment 2comprising a sequence of 10-16 nucleobases shown in SEQ 10 NOS: 1, 16,17, 18, 33, 34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 73, 88, 103, and118 or an allelic variant thereof.
 4. The oligonucleotide according toany one of embodiments 1-3 comprising sequences shown as SEQ ID NOS:1-132.
 5. The oligonucleotide of any one of the preceding embodimentsrepresented by SEQ ID NOS: 133-172.
 6. The oligonucleotide according toanyone of the preceding embodiments, wherein said nucleobase sequencecomprises internucleobase linkages independently selected fromphosphodiester, phosphorothioate and boranophosphate.
 7. Theoligonucleotide of anyone of the preceding embodiments, wherein at leasttwo of said nucleobases are nucleotide analogues.
 8. The oligonucleotideaccording to embodiment 5, wherein said sequence of nucleobasescomprises, in a 5′ to 3′ direction (i) region A: a stretch of 2-4nucleotide analogues, followed by (ii) region B: a stretch of 6-11nucleotides or nucleotide analogues different from those of region A,followed by (iii) region C: a stretch of 2-4 nucleotide analogues, andoptionally followed by (iv) region D: one or two nucleotides.
 9. Theoligonucleotide according to embodiment 8, wherein the region Acomprises at least one phosphodiester linkage between two nucleotideanalogue units and/or or a nucleotide analogue unit and a nucleobaseunit of Region B.
 10. The oligonucleotide according to embodiment 8 orembodiment 9, wherein region C comprises at least one phosphodiesterlinkage between two nucleotide analogue units and/or a nucleotideanalogue unit and a nucleobase unit of Region B.
 11. The oligonucleotideaccording to anyone of embodiments 1 to 6, wherein all theinternucleobase linkages are phosphorothioate.
 12. The oligonucleotideaccording to any one of embodiments 7 to 11, wherein said nucleotideanalogue units are independently selected from 2′-O-alkyl-RNA,2′-amino-DNA, 2′ fluoro-DNA, locked nucleic acid (LNA), arabin nucleicacid (ANA), 2′-fluoro-ANA, RNA, INA and 2′-MOE.
 13. The oligonucleotideaccording to embodiment 12, wherein the nucleotide analogues areindependently selected from 2′-MOE-RNA, 2′-fluoro-DNA, and LNA.
 14. Theoligonucleotide according to embodiment 13, wherein at least one of saidnucleotide analogues is a locked nucleic acid (LNA).
 15. Theoligonucleotide according to embodiment 14, wherein all the nucleotideanalogues are LNA.
 16. The oligonucleotide according to anyone ofembodiments 12 to 15, wherein LNA is selected from beta-D-oxy-LNA,alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA.
 17. A conjugatecomprising an oligonucleotide of any one of the preceding embodimentsand at least one non-nucleotide or non-polynucleotide moiety covalentlyattached to said oligonucleotide.
 18. A conjugate according toembodiment 17, wherein said non-nucleotide or nonpolynucleotide moietyconsists of or comprises a sterol group such as cholesterol.
 19. Apharmaceutical composition comprising an oligonucleotide according toany one of the embodiments 1 to 16 or a conjugate according toembodiment 17 or embodiment 18, and a pharmaceutically acceptablediluent, carrier or adjuvant.
 20. An oligonucleotide or a conjugateaccording to any one of embodiments 1 to 18 for use as a medicament. 21.Use of an oligonucleotide or as conjugate according to any one ofembodiments 1 to 18 for the manufacture of a medicament for thetreatment of abnormal levels of beta-catenin or a disease or conditionfor which down-regulation of beta-catenin expression is indicated. 22.The use according to embodiment 21, wherein said disease or condition iscancer.
 23. A method of treating a subject suffering from cancer, themethod comprising the step of administering a pharmaceuticalcomposition, oligonucleotide or conjugate according to anyone ofembodiments 1 to 19 to the subject in need thereof.
 24. The use or themethod of anyone of embodiments 21-23, wherein the cancer is selectedfrom colorectal cancer, hepatocellular cancer, endometrial cancer,malignant melanoma, cancer of the ovary, pancreas, pituitary,oesophagus, lung, breast, kidney, haematopoetic system, cervix, CNS,bone, bilial tract and adrenal gland.
 25. A target sequence within thebeta-catenin gene, wherein an antisense oligonucleotide corresponding tosaid target sequence is capable of down-regulating the expression ofbeta-catenin.
 26. The target sequence of embodiment 25, wherein thetarget sequence is selected from the regions of the beta-catenin genecomplementary to SEQ ID NOs 1-132 or allelic variants thereof.
 27. Amethod of down-regulating the expression of beta-catenin in a cell,comprising contacting the cell with an oligonucleotide according toanyone of embodiments 1-16.